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Am J Physiol Heart Circ Physiol 278: H2134-H2142, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 6, H2134-H2142, June 2000

Regulation of types I and III NOS in ovine uterine arteries by daily and acute estrogen exposure

Walid A. Salhab, Philip W. Shaul, Blair E. Cox, and Charles R. Rosenfeld

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75235


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide contributes to estrogen-mediated uterine vasodilation; however, the nitric oxide synthases (NOS) involved and their location within uterine arteries are incompletely documented. We investigated the effects of repetitive daily and acute estradiol-17beta (E2beta ) exposure on uterine hemodynamics and NOS abundance and localization in uterine arteries from nonpregnant ovariectomized ewes receiving daily intravenous E2beta (1 µg/kg, n = 5) or no E2beta (n = 7) for 5 days to determine NOS abundance, cGMP contents, and NOS immunohistochemistry. Daily E2beta increased basal and E2beta -mediated rises in uterine blood flow (UBF) 36 and 43% (<0.01), respectively, calcium-dependent NOS activity 150% (P < 0.02) in endothelium-intact and -denuded (~40% of total NOS) arteries, and cGMP contents 39% (P < 0.05). Endothelial (eNOS) was detected in luminal endothelium, whereas neuronal NOS (nNOS) protein was only in the media. A second group of ewes received E2beta (1 µg/kg iv) for 4 days and acute intravenous E2beta (n = 8) or vehicle (n = 4) on day 5. UBF rose 5.5-fold (P < 0.001) 115 min after E2beta , at which time only endothelium-derived calcium-dependent NOS activity increased 30 ± 13% (P < 0.05). Daily E2beta enhances basal and E2beta -mediated increases in UBF, which parallel increases in endothelium-derived eNOS and smooth muscle-derived nNOS. Acute E2beta , however, selectively increases endothelium-derived eNOS.

endothelium; vascular smooth muscle; guanylyl cyclase; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE UTERUS IS a unique organ in mammalian species and plays a pivotal role in reproductive physiology. Of particular interest are the mechanisms responsible for the dramatic increases in uterine blood flow (UBF) that occur during the menstrual cycle and pregnancy [blood flow increasing >5- and >35-fold, respectively (32)]. Available evidence suggests that estrogens may modulate these increases in UBF and parallel decreases in peripheral resistance (10, 12, 19, 32). Markee (19) first demonstrated that estrogen was a uterine vasodilator, but it was 40 years later that Killam et al. (12), studying chronically instrumented nonpregnant ewes remote from anesthesia and surgical stresses, demonstrated the potency of estrogen as a vasodilator. These investigators reported a >10-fold rise in UBF within 90-120 min after an intravenous bolus of estradiol-17beta (E2beta , 1 µg/kg) vs. the 50-100% increases previously observed (10). Rosenfeld and colleagues (18, 34) confirmed these observations using radionucleide-labeled microspheres and subsequently demonstrated that uterine responses to E2beta are locally mediated and independent of its systemic vasodilatory effects. Similar responses to estrogen have been described in women (27, 29). Thus estrogen is a potent vasodilator of reproductive and nonreproductive tissue in several species (32, 34), which may also play an important role in preventing cardiovascular disease in women (20).

Although the mechanism(s) responsible for estrogen-induced vasodilation have been extensively studied, they remain unclear to date (20). The uterus is extremely responsive to the vasodilating effects of E2beta and can be studied in isolation in vivo (12, 32, 34). Thus it is an excellent organ in which to investigate the mechanisms responsible for E2beta -induced vasodilation. Several agents infused directly into the uterine circulation increase UBF, but none demonstrates either the pattern or the persistence of response seen with E2beta (31, 32). In nonpregnant ewes, uterine and systemic responses are reproducible and are characterized by a 30-min delay followed by a peak and plateau at 90-120 min with a gradual decline over 8-12 h (12, 32). This response is reversibly inhibited by cycloheximide (12) but is unaffected by pretreatment with actinomycin D (26, 30), suggesting new protein synthesis is involved and that translational or posttranslational events may be important.

Nitric oxide (NO), a potent vasorelaxant produced from the conversion of L-arginine to L-citrulline by a family of nitric oxide synthases (NOS; see Refs. 9 and 22), is involved in E2beta -mediated uterine vasodilation (33, 41). The actions of NO are mediated by activation of soluble guanylyl cyclase and consequent increases in smooth muscle cGMP (21). In nonpregnant ewes, E2beta -induced increases in UBF are paralleled by increases in uterine cGMP secretion, and both are inhibited by the NOS antagonist nitro-L-arginine methyl ester (L-NAME; see Ref. 33). It is unclear, however, which NOS isoforms are involved. There are two functional classes of NOS isoforms (22). The "inducible" isoenzyme iNOS (type II) binds calmodulin tightly at resting intracellular calcium concentrations and is considered to be calcium independent. The "constitutive" isoenzymes, endothelial NOS (eNOS, type III) and neuronal NOS (nNOS, type I), reversibly bind calmodulin and are considered calcium dependent (22). Although type III NOS may be involved in the effects of E2beta on the uterine circulation (33, 40-42), it is unclear if type I or type II NOS is involved, if responses by the uterine vasculature to chronic and acute estrogen differ, and if NOS abundance is altered. Therefore, the present experiments were designed to determine 1) whether chronic (i.e., daily) and/or acute E2beta exposure upregulate NOS abundance in the uterine arteries of nonpregnant ewes, 2) what NOS isoforms are expressed in the uterine arteries of nonpregnant ewes in the presence or absence of E2beta and where in the artery they are expressed, and 3) whether daily estrogen replacement resembling that used in women affects uterine artery cGMP contents. We also examined parallel hemodynamic effects of daily E2beta on basal and estrogen-induced increases in UBF.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation. Nonpregnant ewes of mixed Western breed were used in the present studies. The surgical procedures and preparations have been described in detail (12, 18, 33). In brief, each animal was fasted overnight but was allowed access to water. In the morning, animals received intramuscular atropine sulfate, and a percutaneous jugular venous catheter was placed for infusion of anesthetic agents. With the use of intravenous ketamine hydrochloride and pentobarbital sodium anesthesia, animals were ovariectomized, 3.0-3.5 mm (ID) electromagnetic flow probes (Micron Instruments, Los Angeles, CA) were implanted around the middle uterine artery of each uterine horn proximal to the first bifurcation, and polyvinyl catheters containing heparinized saline (250 U/ml) were implanted in a femoral artery and vein to the level of the descending aorta and abdominal vena cava, respectively. Postoperatively, ewes were maintained in large individual stalls within the laboratory and were given standard animal chow and water ad libitum. Studies were not begun until the 4th to 5th postoperative day. The studies described were approved by the Institutional Review Board for Animal Research at the University of Texas Southwestern Medical Center at Dallas.

Experimental protocols. The E2beta (Steraloids, Wilton, NH) was dissolved in 95% ethanol and was stored at 4°C as a stock concentration of 1 mg/ml. This solution was diluted to 100 µg/ml with 95% ethanol and was allowed to reach room temperature before injection. Two protocols were used in the following studies. In the first protocol, we determined the effects of daily intravenous E2beta (1 µg/kg) on basal UBF, the magnitude of acute E2beta -induced increases in UBF after consecutive daily doses rather than a continuous infusion (16), and its effect on uterine artery NOS expression and cGMP content. Animals were randomized into two groups. The experimental group (n = 5) received E2beta (1 µg/kg iv) over 1-2 min each morning starting on postoperative day 4 while continuously monitoring UBF, mean arterial pressure (MAP), and heart rate, beginning 30 min before E2beta infusion and continuing for 120 min. This dose of E2beta elicits a maximum rise in UBF and results in plasma estrogen levels similar to those seen in women on replacement therapy (12, 16, 32, 36). After consecutive maximal and reproducible responses to E2beta were established for 2-3 days, E2beta was administered for an additional 5 days, and animals were killed on day 6 for tissue collection. The control group underwent a similar protocol until maximum rises in UBF were consistently documented for 2-3 days, after which they were free of E2beta for 5 days and were killed on day 6 for tissue collection.

In the second protocol, we examined the effects of acute E2beta treatment on NOS expression in uterine arteries of nonpregnant sheep. All animals received E2beta (1 µg/kg iv) each morning starting on the 4th postoperative day. After a maximal and reproducible response by UBF to E2beta was established on two to three consecutive days as described above, animals were randomized into two groups. The experimental group received E2beta (1 µg/kg iv) an additional 4 days, and the animals were killed on day 5 after establishing maximum increases in UBF 114 ± 1.3 min after E2beta infusion. The control group followed a similar protocol, but on day 5 the animals were killed 116 ± 8 min after receiving an intravenous bolus dose of the vehicle (0.5-0.6 ml of 95% ethanol). Uterine arteries were collected and stored as described below.

Hemodynamic measurements. MAP in the lower abdominal aorta was monitored continuously via a femoral arterial catheter connected to a pressure transducer (type 4-327-0109; Bell and Howell, Pasadena, CA). Heart rate was determined from the phasic signal derived from the arterial pressure monitor. UBF was monitored continuously with square-wave electromagnetic flowmeters (RC-1000; Micron Instruments). The flow probes have a linear response to flows in the range studied and are provided with a flow signal and a zero flow calibration. All measurements were continuously recorded on a six-channel pen recorder (3000; Gould, Cleveland, OH). Uterine vascular resistance (UVR) was calculated as the MAP divided by the UBF.

Tissue preparation. At the times noted above, animals were killed with an intravenous dose of pentobarbital sodium (120 mg/kg). The abdomen was opened, the intact uterus was removed in block, and first- through second-generation uterine arteries were dissected and placed in cold physiological saline solution. While on ice, the adventitia was removed from arteries with sharp dissection, intraluminal blood was expressed, and samples were frozen rapidly in liquid nitrogen and stored at -80°C until the time of assay. A second set of arteries from each ewe was opened, and the endothelium was removed with a soft cotton swab, frozen in liquid nitrogen, and stored at -80°C. Endothelium removal was documented histologically in randomly selected arteries.

NOS activity. At the time of assay, arteries were homogenized on ice in 50 mmol/l Tris buffer (pH 7.4) containing 2 µg/ml pepstatin A, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 40 µg/ml Nalpha -p-tosyl-L-lysine chloromethyl ketone, 3 mmol/l dithiothreitol, 10 mmol/l 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mmol/l phenylmethylsulfonyl fluoride, and 20 µmol/l tetrahydrobiopterin. The homogenate was centrifuged at 4°C for 15 min at 12,500 rpm. NOS activity in the supernatant was determined by measuring the conversion of L-[3H]arginine to L-[3H]citrulline (4, 15). Briefly, 50 µl of supernatant were added to 50 µl of buffer, yielding final concentrations of reagents as follows: 2 mmol/l beta -NADPH, 2 µmol/l tetrahydrobiopterin, 10 µmol/l FAD, 10 µmol/l flavin mononucleotide, 2.5 mmol/l CaCl2, 15 nmol/l calmodulin, 2 µmol/l cold L-arginine, and 2.0 µCi/ml L-[3H]arginine. After incubation at 37°C for 60 min, the assay was terminated by addition of 400 µl of 40 mmol/l HEPES buffer (pH 5.5) with 2 mmol/l EDTA and 2 mmol/l EGTA. The terminated reactions were applied to 1-ml columns of Dowex AG50WX-8 (Tris form) and were eluted with 1 ml of the 40 mmol/l HEPES buffer. L-[3H]citrulline generated was collected in scintillation vials and quantified by liquid scintillation spectroscopy. In prior studies, we determined that NOS activity measured under the above conditions of excess substrates and cofactors reflects enzyme abundance (15).

In preliminary experiments, we determined that NOS activity was localized to the supernatant and was linear with mass and time for up to 90 min; further assays were performed over 60 min. The specificity of the measured activity was determined using 4.0 mmol/l L-NAME, a NOS-specific antagonist. Calcium dependence of NOS activity was determined by addition of 12.5 mmol/l EDTA. The protein content of samples was measured using the method of Bradford (3) and using BSA as the standard.

NOS immunohistochemistry. Because all detectable NOS activity was calcium dependent (see RESULTS), we performed immunohistochemistry using antisera to either eNOS or nNOS on randomly selected uterine arteries from control and E2beta -treated ewes to identify and localize the sites of NOS expression. For nNOS, uterine arteries were collected at death, fixed with 2% paraformaldehyde in PBS for 4 h at 4°C, immersed in an increasing sucrose-PBS gradient at 4°C (10% sucrose for 90 min, 15% for 60 min, 20% for 60 min), fixed further for 2 h in 10% neutral buffered Formalin, and embedded in paraffin. Sections (4 µm) were mounted on superfrost positively charged slides and allowed to air-dry for >24 h at room temperature. Sections were deparaffinized with xylene and rehydrated using an ethanol-to-water gradient. After being hydrated with PBS and incubated with Protein Blocking Agent (Immunon, Detroit, MI) for 30 min at room temperature, sections were incubated overnight at 4°C with either 1:1,500 nNOS polyclonal antibody or its diluent. After endogenous peroxidases were quenched with 3% H2O2 in H2O for 30 min at room temperature, immunostaining was performed using standard strepavidin-biotin-horseradish peroxidase detection methodology and hematoxylin counterstaining. The specificity of the nNOS antiserum has been published (37). Sections of ovine cerebellum were run concurrently as positive controls. The nNOS antiserum was the kind gift of Dr. Kim Lau (Dept. of Physiology, University of Texas Southwestern Medical Center, Dallas, TX).

To examine immunostaining for eNOS, uterine arteries with and without endothelium were collected, frozen in liquid nitrogen, and stored at -80°C. At the time of immunostaining, the arteries were thawed briefly at room temperature, embedded in an optimal cutting temperature compound (Tissue Tek, Torrance, CA), and rapidly cooled to -20°C. Cryostat sections (4 µm) were thaw mounted on positively charged slides and immediately fixed in 2% paraformaldehyde for 10 min at 4°C. After being hydrated with PBS and incubated with Protein Blocking Agent for 30 min, sections were incubated overnight at 4°C with either 1:200 eNOS monoclonal antibody (Transduction Laboratories, Lexington, KY) or its diluent. Further processing was as described for nNOS. Sections of ovine pulmonary artery endothelial cells were run concurrently as positive controls for eNOS (37).

Because immunostaining for nNOS was observed in vascular smooth muscle (see RESULTS), additional consecutive sections from other samples of uterine arteries from E2beta -treated ewes were used to determine the presence and/or colocalization of nerve endings and smooth muscle nNOS. Samples were collected and fixed as described above. Consecutive 3-µm sections were mounted on positively charged slides as described and incubated in unlabeled blocking antibody solution for 25 min and then for 25 min with either 1:2,000 neuronal polyclonal antibody (S-100; Dako, Carpenteria, CA), 1:1,000 nNOS antibody, or diluent. Further processing was as described for nNOS. Sections of ovine small bowel and human cerebellum were run as positive controls for nerve endings and nNOS.

Measurement of cGMP. The cGMP content of endothelium-denuded uterine arteries harvested from E2beta -treated and E2beta -free animals was measured by RIA (New England Nuclear, Boston, MA). Briefly, 50 mg of frozen uterine artery were homogenized in 1 ml of 6% TCA and centrifuged at 3,000 g to remove bulk proteins. TCA was then extracted from the resultant supernatant with water-saturated dimethyl ether (3 times). The supernatant was precipitated by lyophilization, dissolved in 0.05 M sodium acetate, and used in the assay. All samples were studied in a single assay.

Statistical analysis. Paired t-test or Wilcoxon signed-rank sum test, when normality failed, was used for comparison of changes over time. Student's t-test or the Mann-Whitney rank test, when normality failed, was used for comparison of means or medians. Data are presented as the means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Responses to daily E2beta . All animals were estrogen free until 4 days postoperative, when they received their first dose of E2beta (1 µg/kg iv). On that day (early, Table 1) UBF rose 3.9-fold (P < 0.001) 120 min after E2beta administration, and UVR fell. As anticipated (12, 18), MAP was unaffected, whereas heart rate rose 17%. After 4 days of daily E2beta administration (1 µg/kg; late, Table 1), baseline UBF was 36% higher compared with day 1 (P < 0.01), and UVR fell proportionally (P < 0.005). However, MAP and heart rate were unaffected. At this time, the maximum UBF achieved after acute E2beta exposure was 43% greater than that seen on days 1-2 (P < 0.005), whereas MAP was unaffected and heart rate increased 23%, resembling early systemic responses.

                              
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  		Table 1.   Basal hemodynamic measurements and responses to acute E2beta treatment before and after 4 days of daily E2beta in nonpregnant ewes

After reproducible acute increases in UBF after intravenous E2beta were established, animals were randomized to either daily E2beta (n = 5) or to a control group (no E2beta , n = 7) and were killed 6 days later for tissue measurements of NOS activity. Compared with the control group, E2beta treatment resulted in a 150% increase (P < 0.01) in NOS activity in endothelium-intact uterine arteries (Fig. 1A). Because the assay is conducted using excess substrates and cofactors and NOS activity is linear with time under the conditions of the experiment, this reflects a 2.5-fold increase in the abundance of NOS enzyme. To determine the site of NOS enzyme, similar measurements were performed using endothelium-denuded uterine arteries from randomly selected E2beta -treated (n = 2) and control (n = 3) animals. As in intact arteries, NOS activity was present in endothelium-denuded arteries, and values increased 150% (P < 0.02) in treated vs. control tissues (Fig. 1B); thus NOS abundance was increased 2.5-fold. From these measurements, we calculated that ~60% of the NOS activity was localized to the luminal endothelium in both the E2beta -treated and control tissues, whereas the remaining activity was in the smooth muscle and/or adventitia.


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  		Fig. 1.   Nitric oxide synthase (NOS) activity in endothelium-intact (A) and -denuded (B) uterine arteries collected from ovariectomized nonpregnant ewes after 5 days of estradiol-17beta (E2beta ; 1 µg · kg-1 · day-1). NOS activity is expressed as pmol citrulline (cit) · mg protein (prot)-1 · min-1. Values are means ± SE. * P < 0.02 and Dagger  P < 0.01 vs. control.

To determine if the NOS activity in the uterine arteries was calcium dependent and inhibited by a NOS antagonist, we examined the effects of the calcium chelator EDTA and L-NAME. Both EDTA and L-NAME inhibited basal NOS activity >95% (P < 0.05) in uterine arteries from either E2beta -treated or control animals (Fig. 2), demonstrating the presence of a calcium- and calmodulin-dependent NOS isoform, i.e., either eNOS or nNOS, in both intact and denuded arteries.


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  		Fig. 2.   Effect of the NOS antagonist nitro-L-arginine methyl ester (L-NAME; n = 12) and calcium chelator EDTA (n = 2) on basal NOS activity in E2beta -treated and control animals. Data from both groups were combined since inhibition did not differ. Values are means ± SE. * P < 0.05 vs. basal.

To characterize the type and localization of NOS isoforms expressed within uterine arteries after 5 days of daily E2beta , we performed immunohistochemistry for eNOS and nNOS proteins using endothelium-intact and -denuded arteries. With the use of randomly selected endothelium-intact uterine arteries from E2beta -treated animals, eNOS protein was detected in the luminal endothelium (Fig. 3A). There was no eNOS in the medial smooth muscle; however, immunostaining was present in the endothelium of the vasa vasorum in the peripheral media and remaining adventitia. Immunostaining of endothelium-denuded vessels for eNOS confirmed the absence of luminal endothelium in the uterine artery and eNOS in medial smooth muscle (Fig. 3B); immunostaining of the vasa vasorum resembled that in endothelium-intact arteries. In contrast to that seen with eNOS, there was no nNOS immunostaining in the endothelium of the uterine artery or vasa vasorum in endothelium-intact arteries (Fig. 3, C and D). However, there was intense nNOS immunostaining in medial smooth muscle cells that was greater in abluminal vs. luminal smooth muscle (Fig. 3D). Immunostaining was not detected in tissues incubated with buffer (Fig. 3E). Concurrently run positive control preparations of ovine pulmonary endothelial cells for eNOS and ovine cerebellum for nNOS showed abundant immunoreactivity for these markers (data not shown). Immunostaining for eNOS and nNOS in uterine arteries selected randomly from control ewes demonstrated a similar distribution (data not shown).


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  		Fig. 3.   Immunohistochemistry for endothelial NOS (eNOS) and neuronal NOS (nNOS) in uterine arteries from nonpregnant ewes after treatment with daily E2beta (1 µg/kg iv). A: immunostaining for eNOS was observed in the luminal endothelium of the uterine artery and adventitial vessels (arrows; magnification ×20). m, Smooth muscle (media); a, adventitia. B: in endothelium-denuded arteries, immunostaining for eNOS was only present in the adventitial vessels (arrows; magnification ×20). C and D: to improve localization and recognition of vascular morphology and nNOS immunostaining, magnification is ×40. Immunostaining for nNOS was not evident in either the luminal endothelium (C, arrow) or medial vasa vasorum (C and D, arrowheads) of uterine arteries. However, intense nNOS immunostaining was present in smooth muscle cells situated in the peripheral media (D, open arrowhead). E: uterine arteries incubated in the absence of primary antisera showed no immunostaining (magnification ×40); arrow denotes luminal endothelium. These findings were replicated in studies of tissues from 3 animals.

The finding of nNOS in medial smooth muscle was unanticipated. We therefore performed immunohistochemistry on additional arteries from E2beta -treated ewes to determine if neuronal tissue was associated with nNOS immunostaining. Consecutive sections confirmed the presence of nNOS as described above; but there was no evidence of neuronal tissue within the media or adventitia (Fig. 4A). Concurrently run positive control preparations of ovine small bowel (Fig. 4B) and human cerebellum (Fig. 4C) showed abundant immunostaining for neuronal tissue in lamina propria and neurons, respectively, whereas the latter also was positive for nNOS (data not shown).


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  		Fig. 4.   Immunohistochemistry for neuronal tissue in uterine arteries from nonpregnant ewes after treatment with daily E2beta (1 µg/kg iv). A: absence of immunostaining for neuronal tissue within the uterine artery smooth muscle (m) and adventitia (a); magnification ×40. Arrow, luminal endothelium. B: ovine small bowel is a positive control for ovine peripheral neuronal tissue with immunostaining of neurons (magnification ×20) in the lamina propria (arrowheads). C: positive control using human cerebellum; intense immunostaining for neuronal tissue (arrowheads) demonstrating that the S-100 antisera works equally well in ovine and human tissues (magnification ×20).

NO is believed to act through stimulation of smooth muscle guanylyl cyclase and enhanced cGMP production (21). We therefore measured cGMP contents in uterine arteries collected from ewes after daily E2beta treatment (n = 5) and in control animals (n = 6). Only endothelium-denuded uterine arteries were assayed, since cGMP production occurs in the smooth muscle (21). Because of insufficient tissue, one animal was not included in the control group. Daily E2beta increased tissue cGMP contents 39%, with values increasing from 9.7 ± 1.0 to 13.5 ± 1.3 fmol/mg wet wt (P < 0.05).

Responses to acute E2beta exposure. In these studies, we wished to determine if a bolus dose of intravenous E2beta in estrogen-primed ewes further modified NOS abundance in ovine uterine arteries. After maximal and reproducible increases in UBF after E2beta for 6-7 consecutive days were established, animals were randomized to receive either E2beta (1 µg/kg; n = 8) or the ethanol vehicle (n = 4) and were killed immediately after UBF had achieved a maximum rise or at a similar time in control ewes. Acute E2beta treatment increased UBF 457 ± 80% (P < 0.0001) while decreasing UVR 76.4 ± 6.2% (P < 0.003); vehicle had no effect. Compared with arteries from vehicle-treated ewes, there was a 30 ± 13% increase (P < 0.05) in NOS activity/abundance in endothelium-intact arteries from ewes receiving a bolus dose of E2beta (Fig. 5A). In contrast, there was no significant difference in NOS activity in endothelium-denuded uterine arteries from E2beta (n = 4)- and vehicle (n = 2)-treated ewes (Fig. 5B), demonstrating that eNOS in the vasa vasorum and nNOS in the smooth muscle contribute minimally to the rise in NOS activity after acute E2beta exposure. Both L-NAME (n = 5) and EDTA (n = 5) inhibited NOS activity >95% in arteries from both groups, further confirming the presence of only calcium-dependent NOS isoforms (results not shown). As described earlier, ~60% of total calcium-dependent NOS activity in the treated and control tissues was associated with the endothelium.


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  		Fig. 5.   NOS activity in endothelium-intact (A) and -denuded (B) uterine arteries collected from ovariectomized nonpregnant ewes at the time of maximum uterine vascular responses after acute E2beta exposure (1 µg/kg iv) or vehicle (ethanol). NOS activity is expressed as pmol citrulline · mg protein-1 · min-1. Values are means ± SE. * P < 0.05 vs. vehicle.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Increases in blood flow to reproductive and nonreproductive tissues occur after chronic and acute estrogen exposure (16, 17, 34). It is likely that this plays a role in its beneficial effects in women (20, 27, 29) and in decreases in vascular resistance in pregnancy (32). The vascular responses to estrogen and the mechanisms involved may vary with the frequency and type of estrogen exposure, but this is unclear. The ovine uterine vascular bed can be instrumented, and both the uterine and systemic responses to estrogen can be studied in detail (32, 33). NOS contributes to uterine vascular responses to chronic and acute estrogen exposure (33, 40-42), but the isozymes involved are incompletely characterized. Using a protocol resembling that for women receiving oral estrogen replacement (29, 36), we compared the effects of repetitive daily doses and acute estrogen treatment on NOS enzyme activity in uterine arteries and parallel changes in uterine and systemic hemodynamics. After daily E2beta for 5 days, calcium-dependent NOS activity in uterine arteries increased 2.5-fold, whereas calcium-independent NOS activity was not detected. This increase in calcium-dependent NOS activity reflected increases in both luminal NOS activity derived from eNOS and medial smooth muscle NOS activity derived from nNOS, which paralleled modest increases in basal uterine artery cGMP contents and UBF and were associated with enhanced uterine vascular responsiveness to the acute vasodilating properties of E2beta . In contrast, whereas UBF increased more than fivefold 90-120 min after acute E2beta exposure in estrogen-primed ewes, only endothelium-derived NOS activity was increased. Thus daily and acute estrogen exposure increases UBF and calcium-dependent NOS abundance in uterine arteries; however, there is evidence of differential expression and regulation of NOS isoforms within the vessel wall.

Uterine and systemic hemodynamic responses to continuous and acute estrogen infusions are well characterized (16, 18, 32), whereas responses to intermittent daily E2beta treatment are less well described, reflecting the general use of continuous estrogen infusions in most animal models studied. When E2beta is continuously infused for 5-6 days in nonpregnant sheep, MAP and systemic vascular resistance fall and heart rate increases within 24 h; this persists throughout the treatment period (16). Although UBF increases initially, reflecting "acute" responses to E2beta , values return to baseline by 5-7 days (16), and within 24-36 h there is marked refractoriness to the acute vasodilating properties of E2beta (6, 32). In contrast, 5 days of intermittent daily E2beta in nonpregnant ewes affected neither basal MAP nor heart rate, basal UBF was persistently increased, and rapid uterine vascular responses to acute E2beta exposure remained intact while the magnitude of the response increased. Although the maximum UBF achieved was 43% greater after 4-5 days of daily E2beta , basal UBF also rose 36%; thus, the relative rise in response to acute E2beta was similar (~4-fold). Because persistent vasodilation was evident with daily E2beta and heart rate was unaffected, the maintenance of MAP may reflect increases in cardiac output via an increase in stroke volume (16). Postmenopausal women receiving daily oral steroid replacement demonstrate a similar fall in UVR (29), and they maintain arterial pressure by increasing cardiac output (43). Women receiving transdermal estrogen replacement also demonstrate a rise in cardiac output (28), but, in contrast to the ewe, they have a persistent fall in UVR despite having plasma E2beta levels resembling that seen in continuously infused sheep (8, 16). Thus there may be some differences between species in the uterine responses. The absence of vascular refractoriness to the acute vasodilator effects of E2beta seen in the ewe during intermittent daily estrogen exposure supports the suggestion by Clewell et al. (6) that the loss of vascular responsiveness during continuous E2beta is due to binding of available estrogen receptors and a lack of unbound receptors through which to mediate acute responses to estrogen. Although this is plausible, studies of estrogen receptor binding have not been performed.

Initially, eNOS and nNOS were considered constitutive enzymes. It is now clear that both are inducible (5, 15, 39, 44). After 5 days of daily E2beta , calcium-dependent NOS activity increased 2.5-fold in intact uterine arteries, resembling that seen in pulmonary endothelium after 48-96 h of E2beta exposure (15). This NOS activity, however, was clearly derived from both the endothelium and the media, the latter accounting for ~40% of total activity. Moreover, both sources of NOS activity were similarly enhanced by daily E2beta , and the increase in total NOS activity was associated with increases in basal uterine artery cGMP contents and a parallel rise in basal UBF, supporting the conclusion of a causal relationship. Because the presence of a smooth muscle NOS was unanticipated from prior reports (40), we performed extensive immunohistological studies of uterine arteries from E2beta -treated and control ewes to determine the isoforms expressed and their distribution within the arterial wall. There was intense eNOS immunostaining only in the luminal endothelium of the uterine artery and vasa vasorum. In no instance was there eNOS immunostaining in the media, demonstrating specificity in its localization. In contrast, nNOS immunostaining with a specific antisera (37) was only seen in the media and was not evident in the endothelium of any artery, further demonstrating the specificity of this antiserum since we and others (40) have not found calcium-independent NOS in the uterine artery. To rule out any association with neuronal tissue within the uterine artery wall, additional immunostaining was performed using an antiserum specific for neuronal tissue (Fig. 4). Although immunostaining was evident in the myenteric plexus of the ovine lamina propria and the human cerebellum (also positive for nNOS), there was none in the uterine artery wall. Thus these are the first data to show nNOS expression in uterine artery myocytes and to suggest that it is regulated by estrogen.

Recently, Boulanger et al. (2) also observed nNOS in the carotid artery smooth muscle. As in the present study, it accounted for ~40% of the total calcium-dependent NOS activity in intact arteries. Lubarsky et al. (14) suggested that a nonendothelial NOS was present in the hindlimb vasculature of pregnant rats, but they did not identify the isoform or localize it within the vessel wall. Although eNOS and nNOS mRNA are reported to increase in various tissues after estrogen exposure (5, 15, 39, 44), few investigators have examined vascular smooth muscle. Veille et al. (42) reported increased calcium-dependent NOS activity in intact ovine uterine arteries after 3 days of continuous E2beta infusion. They did not note the effects on UBF or uterine artery cGMP, nor did they determine the site of NOS expression within the artery or the isozyme(s) involved. Vagnoni et al. (40) also observed increased eNOS in uterine arteries from nonpregnant ewes after E2beta exposure. Consistent with our observations, they did not detect iNOS, suggesting it is not normally present in uterine arteries and that it is not regulated by estrogen. They do not comment on the presence of a NOS isoform in medial smooth muscle. Thus our data support the thesis that daily E2beta increases eNOS abundance in luminal endothelium and nNOS in medial smooth muscle, which increase basal UBF through NO-mediated increases in smooth muscle cGMP. They also demonstrate that E2beta -induced increases in basal NOS activity within the vessel wall potentiate acute vascular responses to subsequent bolus doses of E2beta and other agonists (1, 6).

Bolus doses of E2beta (1 µg/kg) characteristically increase UBF, with maximum increases occurring at 90-120 min (32). This is associated with parallel increases in uterine cGMP secretion, and both are "partially" inhibited by local L-NAME infusions and are reversed by L-arginine (33, 41). Thus NOS activation contributes to acute E2beta -mediated rises in UBF, but the NOS enzymes involved and cellular mechanisms are unclear. In the present studies, endothelium-derived NOS activity increased ~30% at the time of maximum E2beta -mediated UBF. Although the nonendothelial component of NOS activity accounted for ~40% of total uterine artery activity, unlike that seen with daily E2beta exposure, this component was unaffected by acute E2beta . Thus the eNOS in vasa vasorum and nNOS in the media were unresponsive to acute E2beta exposure, whereas luminal eNOS was increased. Of interest was the contrast between the approximately fivefold rise in UBF and 30% rise in eNOS abundance. This suggests that additional endothelium-independent mechanisms are involved in acute E2beta -induced vasodilation or that existing NOS is also activated. Estrogen alters ionic channel activity (7, 11, 38, 45, 46) and relaxes coronary arteries through endothelium-independent mechanisms involving cGMP (7, 23, 45). In coronary artery smooth muscle, E2beta activates calcium-dependent potassium channels (BKCa) through a cGMP-dependent mechanism involving iNOS (7). However, Vagnoni et al. (40) have not found iNOS in uterine artery smooth muscle. In recently completed studies, Rosenfeld and White (35) have identified BKCa in uterine artery myocytes and have partially inhibited acute uterine vascular responses to E2beta with local tetraethylammonium infusion, a specific inhibitor of BKCa (25). Thus, although uterine artery eNOS abundance increases in luminal endothelium within 2 h after acute E2beta , the present data support prior observations from this laboratory (33) that additional mechanisms are involved.

Steroid hormones have generally been considered to exert their effects through classical receptor-mediated genomic mechanisms. In the present study, daily E2beta appears to exert its effects on the uterine vasculature via classical genomic mechanisms, whereas responses to acute E2beta exposure may reflect a combination of nongenomic and genomic mechanisms, consistent with suggestions by others (20). It is plausible that genomically derived increases in uterine artery type I and III NOS mediate the rise in basal UBF via increases in smooth muscle cGMP. It also could account for the increased vascular sensitivity to acute E2beta . Although we have not proven transcriptional regulation in the endothelium or myocyte, E2beta increases eNOS protein and mRNA in pulmonary endothelium and other tissues by enhancing gene transcription (5, 15, 20, 44). On the other hand, nongenomic effects of E2beta likely account for increases in UBF 30 min after a bolus dose of E2beta , suggesting activation of existing NOS (33). This is consistent with reports that this response is inhibited by cycloheximide but not actinomycin D (12, 26, 30). More recently, we found that cycloheximide inhibits not only increases in UBF but also uterine cGMP production (unpublished results). Further support for a nongenomic effect is obtained from reports that E2beta rapidly increases eNOS activity in pulmonary endothelial cells through a receptor-mediated mechanism (13) as well as responses in other tissues (20, 24). Because NOS abundance increased 2 h post-E2beta treatment, there may have been an increase in NOS transcription, but this was not addressed in the present study. Alternatively, it could reflect posttranscription processes. Nonetheless, only endothelium-derived NOS was increased, suggesting that nNOS may not have a rapid response to E2beta or that the sensitivity of our assay cannot detect this. Additional studies of the uterine vasculature are needed to address these questions.

In the present study, we have demonstrated that vascular responses to E2beta depend on the type and duration of exposure. We also provide evidence that the uterine vascular bed, which can be studied in isolation in intact animals remote from the stresses of surgery, is an excellent model in which to explore the mechanisms whereby estrogen has its vascular effects (32). This is supported by the similarity of the present observations with those in postmenopausal women undergoing daily estrogen replacement (29, 43). We also present for the first time that nNOS (type I) is present in uterine vascular myocytes and that its activity/abundance is regulated by chronic estrogen exposure. Additional studies are needed to elucidate the contribution of each NOS isozyme to the vascular responses to E2beta , to determine what other mechanisms are involved, and to deduce how estrogen modulates NOS activation and abundance.


    ACKNOWLEDGEMENTS

We thank Todd Sherman for performing the immunohistochemistry for NOS isoforms, Tim Roy for performing the in vivo estrogen responses, and Patricia Nuckolls for help in preparing the manuscript.


    FOOTNOTES

These studies were supported by National Institute of Child Health and Human Development Grants HD-08783 and HD-30276.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. R. Rosenfeld, Dept. of Pediatrics, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9063 (E-mail: crosen{at}mednet.swmed.edu).

Received 5 November 1999; accepted in final form 3 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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