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Regular Article |
a Departments of Obstetrics and Gynecology,
b Animal Sciences,
c Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin 53715
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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28% during occlusion. The redistribution of UBF to other reproductive tissues suggests that collateral circulation develops in response to occlusion. Systemic arterial and uterine venous NOx levels were reduced 22.1% ± 6.7% and 22.6% ± 7.6%, respectively, during occlusion. Treatment with microspheres produced an unexpected initial (2.5 h) increase in systemic arterial and uterine venous NOx levels by 116% ± 30% and 97% ± 49%, respectively. Despite a decline in NOx levels after 6 h, no significant differences versus preocclusion NOx levels were detected by 24 h of occlusion in this experimental group. In contrast, NOx, UBF, and uterine perfusion pressure levels unexpectedly failed to return to baseline values following release of occlusion. No differences in uterine artery eNOS expression were demonstrated by Western analysis from occlusion. Thus, our data suggest that shear stress may mediate in vivo vasomotor tone via production of NOx.
nitric oxide, placenta, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clark et al. [9] developed a model for studying acute and chronic reductions of UBF in pregnant sheep, thus allowing for reproducible, stable reductions in UBF in which the level of fetal hypoxia can be carefully controlled. A reduction of UBF by 40%–45% is well within the margin of safety described previously for not altering fetal oxygenation in the sheep [10, 11]. Although this level of UBF reduction does not induce fetal hypoxia, fetal and placental growth is moderately restricted, the degree of restriction depending on the level and duration of UBF reduction [12, 13]. The fetus, however, has the ability to adapt to short-term (24 h) reductions in oxygen delivery by increasing oxygen extraction, which maintains fetal oxygen consumption [11, 12, 14, 15]. Alternatively, the uterus has the potential to develop collateral circulation in response to reduced UBF, which serves as a physiologic adaptation to maintain an adequate supply of blood to the uterus and thus to maintain fetal oxygen delivery [16, 17]. Approximately 85% of UBF is derived from the middle uterine arteries in the sheep. When this source of blood is occluded, the dorsal uterine arteries, ovarian arteries, and small cervical branches become a functionally important source of collateral blood flow to the uterus. Anastomoses are also present in the uterine vasculature; however, they are not normally considered a functionally significant source of collateral flow [16]. Nonetheless, data from human [18] and ovine [19] pregnancy indicate that anastomotic channels increase in size and functional significance after occlusion of major vessels.
Flow (shear stress)-mediated dilation is augmented during pregnancy [20–22]. Both NO [23, 24] and cGMP [23] are elevated by shear stress. Furthermore, a number of investigators reported increased eNOS mRNA and protein expression [25–27] and NOS activity [26, 28] in endothelial cells exposed to elevated mechanical forces. It is not known if reduced UBF, and thus shear stress, will locally downregulate the elevated NO levels and/or eNOS expression in the pregnant ewe.
In the present study, we investigated the hypothesis that reducing UBF and thus shear stress in vivo locally decreases uterine arterial NO production and eNOS protein expression. The specific objectives of this study were to 1) demonstrate that production of NO is partly dependent on UBF, 2) characterize the effects of collateral circulation on the redistribution of UBF during unilateral occlusion of the middle uterine artery, 3) elucidate the role of collateral circulation during occlusion on uterine arterial eNOS protein levels, and 4) confirm the earlier observation of Clark et al. [9] that reduced UBF does not alter systemic and contralateral uterine cardiovascular parameters in the sheep.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research and Animal Care and Use Committees of both the Medical School and the College of Agriculture and Life Sciences and follow the recommendations of the ‘Report of the American Veterinary Medical Association Panel on Euthanasia.’ Pregnant (n = 15; 124 ± 1 days' gestation; term = 145 ± 3 days' gestation) ewes of mixed Western breed were used in the present study. Prior to surgery, the ewes received i.m. ketamine (16 mg/kg), atropine (12 µg/kg), and antibiotics (400 000 U penicillin, 200 mg Gentamicin) as described previously [29]. The abdominal, inguinal, and cervical (n = 7 sheep for left ventricle catheter) regions were shaved and aseptically scrubbed. Under sterile conditions, the uterus was exposed following a midventral laparotomy. Transonic flow probes (6 mm; Transonic Systems Inc., Ithaca, NY) were implanted around the middle uterine artery of each uterine horn. Polyvinyl catheters (Tygon, Cleveland, OH) containing heparinized saline (25 IU/ml) were implanted retrograde into the right and left distal branches of the uterine artery (0.23-mm inner diameter [ID], 0.47-mm outer diameter [OD]) and vein (0.40-mm ID, 0.70-mm OD). Balloon cuff occluders (6–8 mm; Rhodes Medical Instrument, Inc., Woodland Hills, CA) were also implanted 2–4 cm proximal to the flow probes. After closure of the midline incision, a catheter (0.40-mm ID, 0.70-mm ) was inserted into both superficial saphenous femoral arteries and advanced through the femoral circulation into the abdominal aorta (20 cm). In seven sheep, a catheter was also inserted into the left ventricle via the right carotid artery for injections of microspheres. The position of the catheter tip in the left ventricle was confirmed by the characteristic pressure pattern recorded on a computer through a DATAQ (Akron, OH) data acquisition system [29]. All catheters were filled with sterile, heparinized saline (25 IU/ml), sealed, and exteriorized with the flow probe leads to a pouch on the ewe's flank. The ewe was allowed access to food and water ad libitum and given i.m. antibiotic (400 000 U penicillin, 200 mg Gentamicin) every other day thereafter and i.m. analgesia (75 mg Flunixin) 24 and 48 h postsurgery. Experimental protocols were begun 8–10 days postoperatively.
Experimental Protocol I
Thirty minutes prior to occlusion of one of the randomly selected middle uterine arteries (n = 8; 125 ± 1 days' gestation), control blood samples and continuous recordings of systemic blood pressure, uterine artery perfusion pressure, heart rate, and UBF were initiated as described previously [29]. When the number of fetuses and side of uterus occluded (gravid vs. nongravid) was accounted for, we noticed that there were differences in the degree of NO2/NO3 (NOx) reduction in response to occlusion. This discrepancy was not discovered until the conclusion of this experiment and is considered to be a major source of variability in our results. The ipsilateral uterine artery was manually occluded by filling the balloon cuff with sterile water and clamping the catheter extension to establish a steady-state, unilateral 40% reduction in UBF [9, 12, 13]. This level of occlusion was carefully chosen to prevent fetal hypoxia. The occlusion was maintained and adjusted if necessary to maintain 40% UBF reduction for 24 h, and blood samples were simultaneously obtained from the uterine venous and systemic arterial catheters at 0.25, 0.5, 1.5, 2.5, 3.5, 5.5, 23.5, and 24 h. The blood was placed in ice-cold tubes containing potassium EDTA until further processing. The occlusion was released after 24 h in four sheep, and blood samples were collected at 2-h intervals thereafter until 28 h. In four other sheep, the occlusion was maintained for 24 h, and the ewe was killed with pentobarbital sodium (50–70 mg/kg), and endothelial-isolated protein preparations were collected from the ipsilateral and contralateral uterine arteries pre- and postocclusion, ovarian artery, and the paracervical artery to assess the role of collateral circulation using Western analysis for eNOS levels.
Experimental Protocol II
This protocol is the same as in protocol I except radioactive microspheres were infused via the left ventricle for measurements of regional tissue blood flows (n = 7; 123 ± 2 days' gestation). One minor modification in this protocol is that blood samples were obtained at 18.5 h instead of at 23.5 h.
Radioactive Microspheres for Measurement of Tissue Blood Flows
The procedure for microspheres was described previously [29]. Briefly, a control injection (preocclusion) of randomly selected radioactively labeled (153Gd, 51Cr, 113Sn, 85Sr, 95Nb, 46Sc) microspheres (3–5 million) were administered into a left ventricular catheter while the appropriate reference blood samples were withdrawn at a rate of 4.12 ml/min from the femoral arterial catheter. Additional randomly selected microsphere isotopes were injected at 3, 6, and 24 h of occlusion and at 1 and 3 h postocclusion. The times of microsphere injections were based on cardiovascular profiles obtained in experimental protocol I. Repeated injection of various microsphere isotopes can be performed in the same animal because they are effectively trapped in the tissue and are not in high enough numbers to induce tissue hypoxia [19, 29, 30]. At the conclusion of the experiment, the animals were killed with an overdose of pentobarbital sodium (50–70 mg/kg), and tissues were obtained, weighed, and placed in wide counting vials for gamma counting (Nuclear Chicago, Des Plaines, IL). The reproductive tissues obtained were the uterus, broad ligament, oviducts, cervix, vagina, vulva, bladder, ovaries, corpus luteum, and mammary gland. Distribution of blood flow within the uterus (endometrium, myometrium, and caruncles/cotyledons) was assessed. The fetal portion of the cotyledon was removed prior to gamma counting of the sample. Regional blood flows were calculated for each organ by the following equation: withdrawal rate divided by the number of microspheres in the reference blood multiplied by the number of microspheres in the tissue samples.
NOx Analysis
Blood samples obtained from each ewe were immediately spun (13 000 x g; 4°C; 10 min), and the plasma was stored at -20°C. At the time of NOx analysis, 500 µl of plasma was added to 1 ml ice-cold 100% EtOH, vortexed, and refrigerated for 30 min at 4°C. The sample was then centrifuged at 12 000 x g for 5 min, and the supernatant was collected for NOx analysis.
NO was measured using chemiluminescence methods as described in our laboratory [31]. Briefly, 100 µl of sample was injected into a gas purge vessel containing 5 ml of vanadium (III) chloride to react and reduce the nitrite/nitrate in the sample back to NO. The gas purge vessel was bubbled with nitrogen to allow NO to be drawn into the NO analyzer (Model 280; Sievers Instruments, Boulder, CO) and mixed with ozone in front of a cooled, red-sensitive photomultiplier tube. Signals from the detector were analyzed by an on-line computer as area under the peak and expressed as mV sec. The measurements reflected the combined concentrations of nitrite and nitrate (NOx) of each sample, which was calculated from a standard curve of 10–2000 pmol nitrates run in each assay.
Procurement of Tissues for Western Analysis
Briefly, the endothelium/tunica intima was gently scraped (4 to 6 times) from the artery using a curved-end spatula and placed directly in lysis buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-100, 0.5% NP-40, 50 mM NaF, and deionzied H2O, pH 7.4, plus the addition of 1 mM Na3VO4, 1 mM PMSF, and 1x proteinase inhibitor cocktail; all from Sigma Chemical Co., St. Louis, MO). We have previously reported and validated this method of isolating endothelial protein to be relatively free of vascular smooth muscle contamination [2]. The endothelium-isolated proteins (endo) were snap frozen immediately upon collection in liquid nitrogen and were stored at -20°C until Western analysis.
Western Analysis of Endo-Isolated Proteins
The protocol for Western immunoblot analysis is essentially as described previously [2, 3, 31] with a few minor modifications. Briefly, endothelial-isolated proteins were solubilized in lysis buffer by sonication. Solubilized protein was then quantified by using a modified Lowry assay procedure (Bio-Rad, Hercules, CA). Proteins (20 µg/lane) were separated by size on 10%–20% gradient polyacrylamide gels (100 V, 1.5 h, MiniProtean II, Bio-Rad) before transfer to Immobilon P membranes (100 V, 2 h). Each blot included a human microvascular endothelial cell (HMEC) lysate (5 µl), developed in our laboratory, as positive control/standard and Rainbow molecular weight markers (Amersham, Arlington Heights, IL). The membrane was probed for eNOS and visualized using enhanced chemiluminescence (ECL) described by Amersham and exposure to Hyperfilm for 10 min. The primary mouse anti-eNOS monoclonal antibody (Transduction Laboratories, Lexington, KY; 1:750 dilution) was directed against sheep anti-mouse serum (Amersham; 1:3000 dilution). Densitometric analysis was performed, and the final data are presented as arbitrary units expressed as a percentage of the ipsilateral preocclusion uterine artery.
Statistical Analysis
Differences among treatment groups were analyzed by Student t-test or repeated measures one-way ANOVA (Sigma Stat; Jandel Scientific, San Rafael, CA) following Bonferroni post hoc test as appropriate. Data are presented as the mean ± SEM.
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RESULTS |
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The distribution of UBF therefore was assessed using the microsphere technique. Throughout 24 h of occlusion, ipsilateral UBF was reduced by 27.7% ± 10.9% (P < 0.03), consistent with the overall trend of the Transonic flow probe measurements, which measures only 80%–85% of total UBF through the middle UA. In contrast, contralateral UBF was unaltered throughout occlusion (Table 2). Because UBF through the middle UA does not account for total UBF, we evaluated tissue-specific redistribution. Partially occluding the middle uterine artery resulted in tissue-specific redistribution of UBF in the ipsilateral caruncles at 6 h (P < 0.10). A similar trend for reduced blood flow to both ipsilateral and contralateral endometrial segments was observed throughout occlusion but did not reach statistical significance. In contrast, no redistribution of myometrial blood flow on either side was observed (Table 2). Within an hour following release of occlusion, an unexpected, transient drop in ipsilateral and contralateral myometrial and endometrial blood flow was observed (Table 2), suggesting that an unknown vasodilatory substance(s) may be contributing to their flow while the occluder was inflated.
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The distribution of total reproductive blood flow using the microsphere technique was also evaluated (Table 2). As expected, blood flow did not change in any systemic vascular bed during occlusion (data not shown). Besides the ipsilateral caruncles, the only other major change was noted in the mammary gland. Unexpectedly, mammary blood flow increased substantially within 3 h of occlusion (P < 0.05) but returned to baseline levels by 6 h. Furthermore, these data suggest the involvement of other vascular beds, including the cervix (P < 0.10) and vagina (P < 0.10), which may play a role in collateral circulation, but further studies are needed to address this issue.
The relative changes in NOx concentrations were evaluated with and without microspheres. Following 24 h of partial uterine arterial occlusion without microspheres, NOx concentrations declined 22.1% ± 6.7% in systemic arterial and 22.6% ± 7.6% in ipsilateral uterine venous plasma compared with control (P < 0.05). In two experiments, a similar but nonsignificant trend was noted for the contralateral uterine venous plasma NOx levels (data not shown). In our initial experiments, we unexpectedly experienced a few technical problems, including failed catheters on one side. Thus, the catheter that still functioned properly was designated ipsilateral (nonrandomly), thus offering an explanation for the low n-value for the contralateral side. Surprisingly, NOx levels failed to return to baseline following release of occlusion in all treatment groups (Fig. 1). It is possible that a reduction in UAP might result in a passive increase in UVR because increases in UVR can be attributed to a decrease in NO production. However, we did not attempt to determine if UAP, in addition to UBF, plays a role in the process.
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In another set of experiments using microspheres, an initial increase in NOx levels preceded the fall in the systemic arterial and the ipsilateral and contralateral venous plasma (Fig. 2). The fall in NOx levels by 24 h did not reach statistical significance in any treatment group studied (P > 0.05). To further address the confounding issue of microspheres on NOx levels, the responses at 2.5 h with (n = 7) and without (n = 7) microspheres was compared. Interestingly, at 2.5 h, a 97% ± 49% and 116% ± 30% increase in NOx levels were observed (P < 0.03) for the ipsilateral uterine venous and systemic arterial plasma, respectively. Alternatively, in singleton pregnancies, NOx levels declined the most when the occluded vessel was on the gravid (n = 6) horn; however, this fall in NOx was less pronounced when the occluded vessel was on the nongravid (n = 4) side, although this difference did not reach statistical significance. In contrast, in ewes carrying multiple fetuses (twins [n = 2] or triplets [n = 1]) in which both horns were gravid, NOx levels also fell to a much lesser degree relative to the singleton pregnancies regardless of side occluded.
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To delineate the effect of partial uterine arterial occlusion on relative eNOS protein expression and collateral circulation, Western analysis was used (Fig. 3). No statistical differences were noted comparing ipsilateral with contralateral UA before (pre-occ) or after (post-occ) occlusion (P > 0.05). Two possible routes of collateral circulation, namely the uterine branch of the ovarian artery and the paracervical artery, were ruled out. No statistical differences were noted between the ipsilateral and contralateral ovarian or paracervical arteries (P > 0.05). This is in agreement with the microsphere data, which showed no differences in ovarian blood flow (Table 2). In contrast with the Western analysis, cervical and vaginal blood flows, as assessed using microspheres, increased acutely at 3 h; however, blood flow returned to baseline within 24 h during occlusion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The microsphere data indicated that blood flow in the caruncles and possibly the endometrium was redistributed during occlusion of UBF. Besides the mammary gland, vagina, and cervix, blood flow did not increase in any other reproductive tissue, suggesting development of collateral circulation. In support of the microsphere data, eNOS protein levels did not change in the ovarian artery or the paracervical artery, two possible sources of collateral flow. Thus, the contribution of collateral flow may be minimal in this experiment; however, we are unable to exclude other possible routes of collateral circulation that may have developed in response to UBF occlusion. Alternatively, the increased blood flow in the collateral circulation may be insufficient to increase eNOS. Other investigators used latex injection and acrylic casts to demonstrate collateral circulation in response to more chronic reductions of UBF [16, 17]. Clapp et al. [19] also demonstrated a 10% redistribution of flow through the embolized main uterine artery to tissues on the contralateral side after 10 days of repetitive unilateral microsphere embolization of the uterine vasculature. Anastomotic channels increase in both size and functional significance after occlusion of major vessels [18, 19, 30]. Thus, increased collateral blood flow may partly represent a physiologic adaptation to maintain an adequate supply of blood to the uterus and thus to maintain fetal oxygen delivery. It was previously proposed that uterine tissues with dual blood supply from the external iliac artery and the internal pudendal artery provide the means by which collateral circulation develops [17]. Mammary blood flow increased 208% ± 117% by 3 h of occlusion, providing a possible route for this collateral circulation. Blood flow to the vagina and cervix also increased at 3 h. In contrast, eNOS protein expression in the paracervical artery was unaltered during occlusion and the rear leg skeletal muscle ipsilateral to the occlusion (data not shown) did not exhibit an increase in blood flow.
Shear stress fell proportionally to the degree of occlusion used in this experiment. We estimated shear stress from our laboratory values using the formula 4
Q/
r3, where is viscosity in Poise, Q is flow through the lumen in ml/sec, and r is the internal radius of the main uterine artery in cm (averaged 0.194 cm for both sides prior to occlusion). Using this formula, shear stress fell distal to occlusion from 59.5 dynes/cm2 to 34.8 dynes/cm2 during partial unilateral UA occlusion, a 41.5% reduction. Contralateral UA shear stress (55.6 dynes/cm2) did not differ from ipsilateral shear stress prior to occlusion and remained stable throughout the experiment. Since lumen diameter, and thus r, was not measured proximal to occlusion, we were unable to estimate shear stress near the occluder. Because increasing shear stress is a potent stimulator of NO production [23, 24], we evaluated the levels of plasma NOx. We demonstrated a reduction in NOx concentrations by 22.1% ± 6.7% and 22.6% ± 7.6% in the systemic arterial and ipsilateral uterine venous plasma, respectively, as a result of reduced UBF. Langille and O'Donnel [32] first reported that reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Rudic et al. [33] subsequently demonstrated that reduced vessel diameter produced a marked reduction in basal NO synthesis with no change in eNOS mRNA or protein levels. The reason for the unexpected fall in NOx levels in the systemic arterial and contralateral uterine venous plasma is unknown. We did not observe a venous-arterial difference in this study, suggesting that the NO metabolites may not be cleared very rapidly from the circulation. Alternatively, we also cannot fully rule out a reduced systemic source of NO in response to unilateral uterine arterial occlusion.
The observation that NOx did not return to control levels 3 h following release of the occlusion also was unexpected; however, changes in NOx are systemic and only a general indicator of relative NO levels. It is noteworthy that NOx levels returned to baseline 24 h postocclusion. Systemic NOx is also an indicator of the sum total of all NO metabolites from the body, thus redistribution of cardiac output and NOx contributions from various vascular beds likely occurred. In addition, the pregnancy-associated rise in NOx in the rat was attenuated by NOS inhibitors (see [7] for a review), suggesting that changes in NO synthesis in the current study likely occur during uterine artery occlusion.
We unexpectedly observed an acute 116% ± 30% and 97% ± 49% increase in NOx metabolites for the systemic arterial and ipsilateral uterine venous plasma, respectively, in the microsphere-treated sheep at 2.5 h of occlusion. The confounding effects of microspheres in vascular studies have been reported by a number of investigators, including vasodilation [34–37] and decreased resistance [38], which are due to the release of diffusible vasodilator substances induced by the microspheres. For example, a 15-µm polystyrene microsphere injection into the bronchial artery of anesthetized sheep caused a large, transient dose-dependent decrease in bronchial artery resistance [36, 37]. Hori et al. [34, 35] also demonstrated that injection of 15-µm microspheres into the coronary circulation of dogs resulted in sustained coronary vasodilation. In contrast, 15-µm microspheres failed to induce vasodilation in the rat pulmonary circulation; however, these investigators noted that vascular resistance increased proportionally with respect to the number of microspheres injected. It is not known if the increase in NOx elicited vasodilation in this experiment since UBF was continually readjusted to maintain 40% less than baseline levels. Events occurring in nonoccluded vessels are also potential sources of vasodilation after microsphere injection [39, 40], including direct interaction of microspheres on the endothelium and adjacent nonoccluded vessels being subjected to increased flow when a microsphere occludes a small arteriole.
Fetal hypoxia is unlikely to occur in this experiment because UBF was only partially occluded for 24 h. In agreement, another laboratory utilizing a chronic reduced UBF preparation also did not observe fetal hypoxia [13]. Other investigators reported that oxygen consumption is maintained for up to 24 h in the presence of reduced UBF [11, 12, 14, 15]. An increase in oxygen extraction despite the fall in oxygen delivery was cited as the reason for maintaining fetal oxygen consumption. We attempted to measure fetal blood gas levels in several sheep; however, due to catheter failure, fetal pO2 was only obtained from one fetus; this was unaltered by uterine artery occlusion. On the other hand, the margin of safety for fetal oxygenation in the sheep allows the fetus to survive up to a 50% reduction in fetal oxygenation [10, 11]. UBF reductions ranging from 30% to 50% were conducted in the earlier studies [9, 13, 15]. UBF was carefully reduced only 40% in this study to maintain oxygen consumption and to prevent fetal hypoxia. However, this level of uterine artery occlusion may alter other fetal blood gas constituents and the acid-base status of the ewe, which may cause the release of vasoactive substances, including NO.
Ipsilateral and contralateral preocclusion uterine arterial eNOS levels were similar after 24 h of reduced UBF. This lack of change may be due to the fact that changes in NOS activity occur before changes in eNOS expression. Thus, eNOS expression may not always be related to NO production, as we have recently shown [3, 5]. Alternatively lower reductions in flow for longer periods of time may be needed to alter eNOS in vivo expression. Because the half-life of eNOS is also relatively long and elevated to near abundance during pregnancy [2–4], altered UBF may not be the primary mechanism for the control of eNOS expression. Others have reported upregulated [41] and downregulated [42, 43] eNOS expression in various vascular beds in response to hypoxia. Xiao et al. [4] recently demonstrated upregulated uterine, but not systemic, artery eNOS mRNA and protein expression in the pregnant sheep in response to chronic hypoxia. Thus, a lack of change in ipsilateral versus contralateral preocclusion uterine arterial eNOS levels suggest hypoxia did not occur in our model. Alternatively, turbulent flow conditions exist in areas immediately proximal to vascular occlusion (see [44] for a review). Both eNOS mRNA and protein expression are unaffected by turbulent flow [27]. In addition, laminar flow, but not turbulent flow, is associated with upregulation of the eNOS gene [45, 46]. Thus, the lack of change in preocclusion vs. postocclusion uterine artery eNOS in the ipsilateral uterine horn may be due to turbulent flow conditions.
In conclusion, we demonstrated herein that occlusion of UBF, insufficient to induce hypoxia, lowers shear stress and results in a fall in NOx by 24 h. The confounding effects of microsphere-induced release of NO and development of collateral circulation must be accounted for in future studies. Thus, shear stress may be important in mediating in vivo vasomotor tone via production of NO in the uterine circulation.
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FOOTNOTES |
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1 Supported, in part, by National Institutes of Health grants HL49210, HL57653, HD33255, and HD38843. J.M.J. is also a recipient of disability supplement HL49210-S1. This study is in partial fulfillment for the MS degree in the Endocrinology-Reproductive Physiology Training Program (www.erp.wisc.edu). 
2 Correspondence: Ronald R. Magness, Department of Obstetrics and Gynecology, University of Wisconsin, Perinatal Research Laboratories, 7E Meriter Hospital, 202 S. Park St., Madison, WI 53715. FAX: 608 257 1304; rmagness{at}facstaff.wisc.edu
Accepted: February 5, 2002.
Received: September 5, 2001.
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