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Am J Physiol Heart Circ Physiol 283: H2389-H2396, 2002. First published September 12, 2002; doi:10.1152/ajpheart.00184.2002
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Vol. 283, Issue 6, H2389-H2396, December 2002

Acute effects of 17beta -estradiol on femoral veins from adult gonadally intact and ovariectomized female pigs

M. P. Bracamonte1, M. Jayachandran2, K. S. Rud2, and V. M. Miller1,2

1 Departments of Physiology and Biophysics, and 2 Surgery, Mayo Clinic and Foundation, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our experiments were designed to determine the acute effects of 17beta -estradiol on femoral veins from intact and ovariectomized female pigs. Rings of femoral veins with or without endothelium were suspended in organ chambers for measurement of isometric force. Concentration-response curves to 17beta -estradiol (10-9 to 10-5 M) were obtained in veins contracted with prostaglandin F2alpha in the absence and presence of inhibitors of either estrogen receptors (ICI-182780; 10-5 M), nitric oxide synthase [NG-monomethyl-L-arginine (L-NMMA); 10-4 M], soluble guanylate cyclase (1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10-5 M), or potassium channels (tetraethylammonium; 10-2 M). Estrogen receptors were identified with the use of Western blotting and immunostaining in veins of both groups. 17beta -Estradiol caused acute endothelium-dependent relaxations in both groups. Relaxations to 17beta -estradiol were inhibited by L-NMMA and 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but not ICI-182780. Tetraethylammonium inhibited relaxations only in veins with endothelium from intact females. Results indicate that 17beta -estradiol causes acute endothelium-dependent relaxations in femoral veins. The relative contribution of nitric oxide and K+ channels as mechanisms involved in relaxations to 17beta -estradiol in femoral veins is modulated by ovarian status.

endothelium; hyperpolarizing factor; nitric oxide; potassium channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OBSERVATIONAL AND EPIDEMIOLOGICAL studies suggest that oral hormone replacement therapy provides primary prevention of coronary artery disease in postmenopausal women while it also increases the risk of venous thrombosis (5, 20, 23, 40, 49, 54, 55). The reasons for these apparent opposite effects on the arterial and venous systems are not known. Effects of estrogen (17beta -estradiol) on the arterial circulation are well characterized (35). For example, 17beta -estradiol has both genomic and nongenomic effects on arteries. Some of these effects include rapid and sustained production and release of endothelium-derived nitric oxide (NO), changes in activation of K+ and Ca2+ channels, and regulation of intracellular Ca2+ (12, 13, 36, 37, 42, 45, 59). However, it is unknown whether 17beta -estradiol could cause effects in veins as endothelial and vascular smooth muscle cells from veins have estrogen receptors (ER)-alpha (ERalpha ) and -beta (ERbeta ) (22). Information is needed to begin to understand mechanisms of how estrogen treatment increases the risk of venous thrombosis. Therefore, the experiments were designed to determine the acute affects of 17beta -estradiol on femoral veins and whether or not these effects were modulated by the ovarian status of the animal with the use of ovariectomy (by laparoscopy) to mimic menopause. This study fills an important gap in the literature because the acute effects of 17beta -estradiol in arteries are well documented (35), but the effects of 17beta -estradiol on veins are unknown.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Gonadally intact and ovariectomized (Ovx) female Yorkshire pigs (6 mo old, 110-120 kg) were used for these experiments. The external genitalia from gonadally intact females showed changes associated with an estrus cycle. Serum levels of estrogen from gonadally intact females range from 10 to 30 pg/ml and estrogen levels in Ovx females are below the sensitivity level of assay (4, 57). Uterine weight was used as a bioassay for the efficacy of surgery and was significantly less in Ovx females (51.3 ± 7.8 g) compared with gonadally intact females (86.6 ± 12.3 g). All pigs were fed twice a day with Lean Grow (Land O'Lakes Farmland Feed), given free access to water, and were housed at 22°C on a 12:12-h light-dark cycle.

Protocol. Four weeks after ovariectomy, Ovx and age-matched gonadally intact female pigs were anesthetized (ketamine and xylazine, 12 and 8 mg/kg, respectively), and the femoral veins were removed. The veins were placed immediately into cold modified Krebs-Ringer bicarbonate solution of the following composition (in mmol/l): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.026 Ca2+ disodium EDTA, and 11.1 dextrose. After the adventitia was trimmed, the veins were cut into rings ~4-5 mm long. Some rings were stored at -80°C for subsequent Western blotting to evaluate expression of ERs. Other rings were prepared for organ chamber experiments or immunohistochemistry.

Organ chamber experiments. Rings with and without endothelium were studied. The endothelium was removed in some rings by gently rolling them on a watchman's forceps that was inserted into the lumen. Removal of the endothelium was verified by histology at the end of the experiments (hematoxylin and eosin staining). Rings with or without endothelium were suspended between a fixed stirrup and a force transducer for the measurement of isometric force in 10-ml organ chamber baths filled with Krebs-Ringer bicarbonate solution, aerated with 95% O2-5% CO2 (pH 7.4) at 37°C. Each ring was stretched to the optimal point on its length-tension curve as determined by tension developed to norepinephrine (3 × 10-7 M). After a 15-min equilibration period at optimal length (baseline tension), a dose-response curve to 17beta -estradiol (10-9-10-5 M) was obtained from baseline tension (no precontraction). In other experiments, rings with or without endothelium at baseline tension were left untreated or incubated with either ICI-182780 (ER blocker, 10-5 M), NG-monomethyl-L-arginine (L-NMMA, a competitive inhibitor of NO synthase, 10-4 M), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10-5 M, a soluble guanylate cyclase inhibitor), or tetraethylammonium acetate (TEA, 10-2 M, a nonspecific K+ channel blocker at this concentration). After 45 min, the rings were contracted with prostaglandin F2alpha (PGF2alpha , 2 × 10-6 M), and once a stable contraction was reached, cumulative concentration-response curves to 17beta -estradiol (10-9-10-5 M) were obtained. Dose-response curves to 17alpha -estradiol (10-9-10-5 M) also were obtained in untreated rings with endothelium contracted with PGF2alpha (2 × 10-6 M) from gonadally intact female pigs. Only one response to 17beta -estradiol was obtained per ring. Once the inhibitors were added, they remained in contact with the tissue throughout the experiment. PGF2alpha (2 × 10-6 M/l) causes ~30-50% contraction to KCl (60 mM) (29). 17beta -Estradiol and 17alpha -estradiol and ODQ were dissolved in DMSO and further diluted with distilled water. Because 17beta - and 17alpha -estradiol were dissolved in DMSO, a dose-response curve to an equivalent concentration of DMSO, used to dissolve the 17beta - and 17alpha -estradiol concentration, was used as a control.

Drugs and chemicals. PGF2alpha , L-NMMA, ODQ, TEA, DMSO, 17beta - and 17alpha -estradiol, and all components of the Krebs solution were purchased from Sigma (St. Louis, MO). ICI-184780 was purchased from Tocris Cookson (Ballwin, MO). Unless otherwise specified, drugs were prepared daily with distilled water. All drug concentrations were expressed as the final molar (M) concentration in the organ chamber baths.

Immunohistochemistry for ERs. Segments of femoral veins not used in organ chamber experiments were fixed in 10% formalin and embedded in paraffin blocks, sectioned (5 µm), and mounted on glass slides. Slides were deparaffinized, dehydrated, and immersed in 50% methanol containing 3% hydrogen peroxide to block endogenous peroxidase activity. The slides were then rehydrated, and an antigen was retrieved by steaming the slides in EDTA (1 mmol/l, pH 8.0) for 30 min. Nonspecific binding was then blocked by incubation in 5% normal goat or rabbit serum (Vector Laboratories) in 1× PBS for 30 min. Excess serum was blotted off, and slides were incubated overnight at 4°C with either an affinity-purified primary polyclonal rabbit antibody against ER-alpha (1:200 in 1× PBS; Santa Cruz Biotechnology) + 5% normal goat serum or with an affinity-purified primary polyclonal goat antibody against ERbeta (1:200 in 1× PBS; Santa Cruz Biotechnology) + 5% normal rabbit serum. Slides were then washed three times (5 min each) with 1× PBS, and biotinylated secondary goat anti-rabbit or rabbit anti-goat antibodies (1:200, Vector Laboratories) were added for 30 min. After the slides were washed (with 1× PBS, 3 times, 5 min each), peroxidase activity was visualized by using a diaminobenzidine tetrahydrochloride substrate kit (brown staining, Vector Laboratories) for 2 min, followed by hematoxylin counterstaining (for control staining only). Sections of porcine uterus (ER positive), the human breast cancer cell line BT-20 (ER negative, American Type Culture Collection; Rockville, MD), rabbit IgG (1:200 in 1× PBS, Vector Laboratories), goat IgG (1:200 in 1× PBS, Chemicon International), and omission of either ERalpha or ERbeta antibodies were used for control staining.

Western blotting for evaluation of ER expression in femoral veins. Equal weight of frozen veins from gonadally intact and Ovx female pigs were minced with a sterile blade and ground in a mortar or homogenized in 10 vol of lysis buffer (1% SDS, 1 mM sodium orthovanadate, and 10 mM Tris pH 7.4) containing a mixture of protease inhibitors (Sigma). The homogenate was centrifuged at 10,000 revolutions/min for 5 min at 4°C. The supernatant was collected, and protein was concentrated with the use of a Centricon (YM10) Centifugal Filter Device (Amicon Bioseparations). Total protein in the sample was determined by bicinchoninic acid protein assay (BCA-200, Pierce; Rockford, IL) using bovine serum albumin as a standard. The remaining concentrated sample was mixed with an equal volume of 2× electrophoresis sample buffer (1× = 125 mM Tris · HCl pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% beta -mercaptoethanol) and heated at 95°C for 5 min. ERalpha and ERbeta human recombinant proteins (Panvera; Madison, WI) were mixed with electrophoresis sample buffer (without beta -mercaptoethanol). Equal amounts of heated samples (50 µg protein) and 1 µg of human recombinant ERalpha and ERbeta protein were loaded in each lane of 7.5% SDS-PAGE (Ready Gels, Bio-Rad). Separated proteins were transferred onto polyvinylidene difluoride membrane (Bio-Rad) using Trans-Blot SD; semidry transfer cell (Bio-Rad). Protein-transferred membranes were blocked for 1 h with 5% nonfat dry milk (Bio-Rad) dissolved in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol). Membranes were incubated with the following primary antibodies (dilutions in transfer buffer overnight at 4°C): mouse monoclonal ERalpha (1:500 dilution; clone AER311, Upstate Biotechnology) and mouse monoclonal ERbeta antibody (1:500 dilution; clone 9.88, Sigma). Membranes were washed two times in 1× Tris-buffered saline (Bio-Rad) for 5 min each and treated with secondary goat anti-mouse IgG-antibody (50 µl in 10 ml 1× Tris-buffered saline) for 2 h at room temperature. Membranes were washed two times in 1× Tris-buffered saline for 5 min, and protein expressions on membranes were determined by colorimetric method using an Opti-4CN Substrate Kit (Bio-Rad) (24).

Statistical analysis. Results are expressed as means ± SE; n refers to the number of pigs from which the femoral veins were removed. Data from organ chamber experiments are expressed as the percent change in tension from contractions to PGF2alpha . Unpaired Student's t-test (two tailed) was done to test results statistically by either comparing maximal relaxations in rings that did not relax or relaxed slightly to 17beta -estradiol (as ED50 or area under the concentration-response curve could not be determined in those rings) or total area under the concentration-response curve. Statistical significance was accepted when P < 0.05. For two-sided tests with alpha  = 0.05, there was 80% power to detect differences between two means ranging from 1.5 to 2.4 standard deviations. Therefore, differences of 0.8 standard deviations would not be detected.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Responses to 17beta - and 17alpha -estradiol. At baseline tension (i.e., in the absence of precontraction with PGF2alpha ), 17beta -estradiol caused a slight relaxation only in rings with endothelium. These relaxations averaged -0.2 ± 0.04 g (n = 5) in intact and -0.1 ± 0.03 g (n = 4) in Ovx, respectively.

In rings contracted with PGF2alpha (2 × 10-6 M), 17beta -estradiol caused concentration-dependent relaxations only in rings with endothelium (Figs. 1 and 2). These relaxations were acute, occurring within 2 min after the addition of 17beta -estradiol (Fig. 2). No relaxations to concentration-matched DMSO controls were observed (Figs. 1 and 2). Threshold concentrations for relaxations to 17beta -estradiol varied between 1 × 10-8 and 3 × 10-7 M. Maximal relaxations to 17beta -estradiol in veins with endothelium from intact and Ovx females averaged -35.3 ± 9.4% (n = 11) and -47.2 ± 10.2% (n = 9), respectively. Maximal relaxations to 17beta -estradiol in veins without endothelium from intact and Ovx females were -0.8 ± 0.8% (n = 6) and -11.5 ± 4.4% (n = 5), respectively. Contractions to PGF2alpha were similar among groups ranging from 1.3 to 2.1 g in rings with endothelium and 1.4 to 2.1 g in rings without endothelium. In other experiments, 17alpha -estradiol caused similar relaxations of rings with and without endothelium (Fig. 3).


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  		Fig. 1.   Cumulative concentration-response curves to 17beta -estradiol in femoral veins with (A) or without (B) endothelium from sexually mature, gonadally intact and ovariectomized (Ovx, for 4 wk) female pigs. n, Number of animals per group. Relaxations are shown as means ± SE of a percent change in tension from contractions to prostaglandin F2alpha (PGF2alpha ; 2 × 10-6 M). 17beta -Estradiol relaxed significantly only femoral veins with endothelium from intact and Ovx female pigs compared with concentration-matched DMSO controls. Contractions to PGF2alpha were not significantly different in rings with (range, 1.3-2.0 g) or without (range, 1.4-2.1 g) endothelium. *P < 0.05, significant difference in relaxations to 17beta -estradiol compared with DMSO controls; unpaired t-test of maximal relaxations.



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  		Fig. 2.   Representative tracing of responses to 17beta -estradiol in femoral veins with endothelium from gonadally intact and Ovx female pigs. Veins were contracted with PGF2alpha (2 × 10-6 M), and 17beta -estradiol was added once contractions had stabilized. Femoral veins from both groups relaxed acutely and similarly to 17beta -estradiol in the presence of endothelium only.



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  		Fig. 3.   Cumulative concentration-response curves to 17alpha -estradiol in femoral veins with or without endothelium from sexually mature, gonadally intact female pigs. Relaxations are shown as means ± SE of a percent change in tension from contractions to PGF2alpha (2 × 10-6 M). Contractions to PGF2alpha were not significantly different between groups. 17alpha -Estradiol relaxed contracted femoral veins with and without endothelium similarly (unpaired t-test of area under the curve).

Mechanisms of relaxation to 17beta -estradiol. Incubating rings with L-NMMA did not produce contractions in femoral veins from either group at baseline tensions. In the presence of L-NMMA (10-4 M), sensitivity to 17beta -estradiol was decreased significantly in femoral veins with endothelium contracted with PGF2alpha (2 × 10-6 M) from intact and Ovx female pigs (Fig. 4A). Only veins from Ovx females relaxed maximally and were similar to Ovx control rings at the highest 17beta -estradiol concentration (10-5 M).


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  		Fig. 4.   Cumulative concentration-response curves to 17beta -estradiol in femoral veins with endothelium from sexually mature, gonadally intact and Ovx (for 4 wk) female pigs in the absence or presence of NG-monomethyl-L-arginine (L-NMMA) (A; 10-4 M, nitric oxide synthase competitive inhibitor) or tetraethylammonium (TEA) (B; 10-2 M, nonspecific K+ channel blocker at this concentration). Relaxations are shown as means ± SE of a percent change in tension from contractions to PGF2alpha (2 × 10-6 M). Contractions to PGF2alpha (2 × 10-6 M) ranged from 1.4 to 2.8 g and were not significantly different among groups. Relaxations to 17beta -estradiol were significantly inhibited by L-NMMA in veins with endothelium from intact and Ovx female pigs. Maximal relaxations to 17beta -estradiol were inhibited significantly only in rings incubated with L-NMMA from intact females. Relaxations to 17beta -estradiol were inhibited significantly by TEA in veins with endothelium from intact but not from Ovx female pigs. Incubating rings with TEA did not increase contractions to PGF2alpha (2 × 10-6 M). *P < 0.05, significant difference in relaxations to 17beta -estradiol in the presence of L-NMMA or in rings from intact females incubated with TEA; unpaired t-test of area under the curve.

Inhibiting K+ channels with the nonspecific inhibitor TEA (at 10-2 M) also significantly decreased relaxations to 17beta -estradiol but only in rings with endothelium contracted with PGF2alpha (2 × 10-6 M) from gonadally intact female pigs (Fig. 4B).

Inhibiting soluble guanylate cyclase with ODQ (10-5 M) significantly shifted the threshold for relaxation to 17beta -estradiol in rings with endothelium from intact and Ovx females (Fig. 5). In the absence of ODQ, relaxations to 17beta -estradiol reached 20% from the contractions to PGF2alpha between 3 × 10-7 and 10-8 M, whereas in the presence of ODQ, 20% relaxation to 17beta -estradiol was not reached until 3 × 10-6 M in both animal groups.


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  		Fig. 5.   Cumulative concentration-response curves to 17beta -estradiol in femoral veins with endothelium from sexually mature gonadally intact and Ovx (for 4 wk) female pigs in the absence or presence of 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10-5 M), a soluble guanylate cyclase inhibitor. Relaxations are shown as means ± SE of the percent change in tension from contractions to PGF2alpha (2 × 10-6 M). Contractions to PGF2alpha (2 × 10-6 M) ranged from 1.8 to 2.8 g and were not significantly different among groups. Relaxations to 17beta -estradiol were inhibited significantly by ODQ in veins with endothelium from intact and Ovx female pigs. *P < 0.05, significant difference in relaxations to 17beta -estradiol in the presence of ODQ; unpaired t-test of area under the curve.

Role of ERs. Relaxations to 17beta -estradiol in femoral vein rings with endothelium contracted with PGF2alpha (2 × 10-6 M) were not significantly inhibited by the nonselective ER antagonist ICI-182780 (Fig. 6).


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  		Fig. 6.   Cumulative concentration-response curves to 17beta -estradiol in femoral veins with endothelium from sexually mature gonadally intact and Ovx (for 4 wk) female pigs in the absence or presence of ICI-182780 (10-5 M, estrogen receptor blocker). Relaxations are shown as means ± SE of a percent change in tension from contractions to PGF2alpha (2 × 10-6 M). Acute relaxations to 17beta -estradiol were not inhibited significantly by ICI-182780 in veins with endothelium from intact and Ovx female pigs (P > 0.05, area under the curve). Contractions to PGF2alpha (2 × 10-6 M) were not significantly different among groups and ranged from 1.6 to 2.2.

Western blotting using monoclonal antibodies demonstrated both ERalpha and ERbeta expression in femoral veins from gonadally intact and Ovx female veins (Fig. 7). The migrating patterns for ERalpha and ERbeta in femoral veins were similar to the migrating patterns observed in purified protein standards.


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  		Fig. 7.   Representative Western blots using monoclonal antibodies for estrogen receptor-alpha (ERalpha ) (A) and -beta (ERbeta ) (B) in veins from intact and Ovx female pigs. Expression of ERs was not altered significantly in intact and Ovx female pigs. Recombinant human ERalpha and ERbeta proteins were used to eliminate specific cross reactivity of ER antibodies. The monoclonal antibodies used in this study (ERalpha and ERbeta ) do not cross react with each other.

Immunostaining for ERalpha and ERbeta was distributed in all components of the femoral vein wall, including endothelium, smooth muscle, and some cells of the adventitia (Fig. 8, A and B). Control staining of porcine uterus was positive for both receptors (not shown), whereas staining of the human breast cancer cell line BT-20 (not shown) and omission of the primary antibodies in staining of femoral veins (n = 3; Fig. 8 insets) were negative for both receptors.


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  		Fig. 8.   Representative immunohistochemical stainings of femoral veins from Ovx (for 4 wk) female pigs for ERalpha or ERbeta . The presence of ERalpha and ERbeta was determined by using affinity-purified polyclonal rabbit antibodies (for ERalpha ) or polyclonal goat antibodies (for ERbeta ), followed by biotinylated goat anti-rabbit or rabbit anti-goat IgG antibodies, respectively. Rabbit or goat IgG (inset; counterstained with hematoxylin) and omission of ERalpha or ERbeta antibodies were used for control stainings. Components of the vein wall are identified as the endothelium (i), media (m), and adventitia (a). Original magnification shown at ×40.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Results from the present study indicate that 17beta -estradiol over a concentration range used in studies of isolated arteries (16, 19, 50) causes rapid acute endothelium-dependent relaxation in femoral veins from both gonadally intact and Ovx female pigs. In veins, 17beta -estradiol did not cause a direct relaxation of smooth muscle (rings without endothelium). On the contrary, 17beta -estradiol relaxes arterial smooth muscle by activation of K+ channels, inhibition of voltage-gated Ca2+ channels and increases in extrusion of intracellular Ca2+ (21, 25, 42, 59).

Concentrations of 17beta -estradiol that cause relaxation of blood vessels in vitro are higher than circulating concentrations in vivo (1), which may reflect diffusion of the steroid across the vein wall or metabolism of 17beta -estradiol to another active compound such as estrone through the actions of 17beta -hydroxysteroid dehydrogenase (6). Estrone does not cause relaxation of the aorta (17); however, vasoactivity of metabolites of estrogen has not been tested in veins. Alternatively, higher concentrations of exogenous 17beta -estradiol may be needed to displace endogenous metabolites bound to cytosolic ERs.

An unexpected finding in the present study was that 17alpha -estradiol also caused relaxations of veins similar to relaxations observed to 17beta -estradiol. However, unlike 17beta -estradiol, relaxations to 17alpha -estradiol also occurred in rings without endothelium. Responses to 17alpha -estradiol are not without precedent as endothelium-independent relaxations to this isoform of estrogen also have been observed in rat aorta and pig coronary arteries (43, 46). 17alpha -Estradiol binds ERalpha and ERbeta with 58% and 11% efficiency, respectively (27), and inhibits Ca2+ influx in bovine coronary arteries (46). It remains to be determined whether a similar mechanism occurs in veins.

In the present study, relaxations of porcine femoral veins to 17beta -estradiol were not inhibited by ER antagonist, ICI-182780 (10-5 M). ICI-182780 causes acute relaxation of veins (9) and, therefore, may act as a partial agonist for ERs in veins as in bone (48). ICI-182780 inhibits the decline in intracellular Ca2+ stimulated by 17beta -estradiol in freshly isolated coronary artery smooth muscle cells of female pigs (42), has high affinity for ERalpha and ERbeta (27) and binds to an allosteric site on the ER, potentiating [3H]estradiol binding to the receptor (15, 61). More studies are needed to better define binding affinities and efficacy of ICI-182780 for ERs in venous endothelium and smooth muscle.

Both ERalpha and ERbeta were identified by immunostaining and Western blotting in porcine femoral veins. These results are consistent with expression of ERalpha and ERbeta as quantified by mRNA in human veins (22). Staining for ERs was identified in endothelial cells, and cells of the media and adventitia. The distribution of staining for ER is consistent with the functional data of endothelium-dependent responses to 17beta -estradiol and endothelium-independent responses to 17alpha -estradiol. It could not be determined from this study whether 17beta -estradiol binds to a putative plasma membrane receptor (33) and/or other proteins to cause acute, nongenomic relaxations to 17beta -estradiol in femoral veins. Nongenomic actions of estrogen in other tissues include increases in cAMP, mitogen-activated protein kinase activity, phospholipase C (28, 39, 51), and activation of Maxi-K (hSlo) and L-type Ca2+ channels (26, 53).

NO may mediate acute relaxations to 17beta -estradiol in femoral veins, as L-NMMA (10-4 M) significantly inhibited relaxations to estrogen in both groups. Estrogen causes releases NO from arterial endothelial cells (11, 44, 47, 60). Venous endothelial cells also synthesize and release NO, although in lower levels compared with arteries (38).

17beta -Estradiol may cause release of endothelium-derived factors such as NO or C-type natriuretic peptides that activate K+ channels in porcine femoral veins (3, 7, 58) as TEA, the nonselective K+ channel inhibitor at 10-2 M(10), decreased relaxations to 17beta -estradiol. However, hormonal status may modulate K+ channel activity and/or number K+ channels as relaxations to 17beta -estradiol were inhibited by TEA in femoral veins from intact but not from Ovx female pigs. mRNA for a K+ channel is rapidly and reversibly induced during estrus and after estrogen treatment in rat uteri (8, 41). Similar effects have not been investigated in vascular tissue.

Increases in cGMP may be a common pathway by which 17beta -estradiol causes relaxations in femoral veins as relaxations were inhibited by soluble guanylate cyclase inhibitor ODQ (18). NO activates directly K+ channels on smooth muscle cells (7). However, release of NO and activation of K+ channels may not be independent or mutually exclusive events because NO also relaxes veins by increasing cGMP levels (30), which in turn may activate Ca2+ activated K+ channels (31, 52). Cyclic nucleotide-gated channels, protein kinases, and/or phosphodiesterases (32) could be additional cGMP targets. Experiments are needed to determine the effects of 17beta -estradiol on cGMP-mediated vasodilating pathways in venous smooth muscle.

Results from the present study provide information to understand the paradox of the beneficial effect of 17beta -estradiol on the arterial circulation while simultaneously increasing risk of venous thrombosis (2, 14, 20, 23). In 1856, the German physician and scientist Rudolph Virchow (56) proposed that venous thrombosis occurred from the interaction of three factors: changes in the vessel wall, reduction in blood flow (stasis), and coagulability of the blood, a theory known as Virchow's triad. Estrogen replacement therapy affects coagulability of the blood through changes in tissue factor pathway inhibitor and C-reactive protein (34). The present study extends study of effects of 17beta -estradiol to the venous wall. Dilation due to decreases in venous tone may cause local blood stasis that, in combination with changes in coagulation, could facilitate clot formation.

In conclusion, results from this study indicate that 17beta -estradiol acutely relaxes femoral veins from intact and Ovx females in an endothelium-dependent manner. 17alpha -Estradiol also relaxes femoral veins in the presence and absence of endothelium. ERalpha and ERbeta are present in intima, media, and adventitia of femoral veins. Mechanisms of relaxations to 17beta -estradiol in femoral veins include release of NO from the endothelium and activation of K+ channels, the relative contribution of which changes with ovarian status. Decreases in venous tone may contribute to blood stasis, which could, in combination with changes in blood coagulability, contribute to formation of thrombus.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-51736 and the Mayo Graduate School of Medicine.


    FOOTNOTES

Address for reprint requests and other correspondence: V. M. Miller, Dept. of Surgery, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail: miller.virginia{at}mayo.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.00184.2002

Received 4 April 2002; accepted in final form 15 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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2.   Balfour, JA, and Goa KL. Raloxifene. Drugs Aging 12: 335-341, 1998.

3.   Barber, DA, Burnett JC, Jr, and Miller VM. Gender differences in relaxations evoked by C-Type natriuretic peptide in porcine coronary arteries. Endothelium 2: s2, 1995.

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Am J Physiol Heart Circ Physiol 283(6):H2389-H2396
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