INTRODUCTION

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DURING PREGNANCY there are dramatic changes in the maternal cardiovascular system that enable the normal growth and development of a fetus. This is an important physiological adaptation to pregnancy and is associated with a substantial increase in cardiac output and uterine blood flow that is essential for fetal growth. Uterine and systemic vasodilation occurs despite an increase in circulating levels of the vasoconstrictor angiotensin II (ANG II). This suggests a refractoriness to ANG II during pregnancy of the systemic and uterine vasculatures that has been demonstrated in human and animal models (7, 14). ANG II appears to play a role in the regulation of uterine and/or umbilical blood flow during pregnancy because the use of angiotensin-converting enzyme (ACE) inhibitors, which inhibit endogenous ANG II production, is associated with fetal growth restriction, fetal hypoxia, and in some cases fetal death (12). These fetal effects of ACE inhibitors may be in part due to reduced uterine and/or placental blood flow (15).

Two main ANG II receptor subtypes have been described in the literature, type 1 (AT₁) and type 2 (AT₂). The AT₁ receptors are predominantly found in the vascular smooth muscle, adrenal cortex, heart, kidney, liver, and lung. The AT₁ receptor is thought to mediate most of the functions that are generally associated with ANG II, including vasoconstriction, uterine contraction, aldosterone and catecholamine release, water intake, and renal salt and water reabsorption (10). The AT₂ receptor is predominantly found in the uterus, brain, adrenal medulla, and heart, and currently its function is not well understood (6, 10, 21).

Cox et al. (3, 5) have reported that the vast majority (85%) of the ANG II receptors in the ovine and human pregnant and nonpregnant uterine vasculature are the AT₂ subtype. Additionally, Cox and co-workers (4) have recently reported that ANG II has no direct vasoconstrictive actions in the uterus but that uterine vasoconstriction occurs following release of systemic vasoconstrictors. On the basis of the predominance of the AT₂ subtype in the ovine uterine vasculature and the fact that ANG II is a uterine vasoconstrictor in this species, we hypothesized that in nonpregnant sheep ANG II acts locally in the uterine vasculature to stimulate AT₂ receptors and produce vasoconstriction.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Seven healthy nonpregnant ewes of mixed breed, weighing between 55 and 85 kg, underwent a thoracotomy and laparotomy on two separate days to catheterize the uterine and femoral vessels and to place transit-time Doppler flow probes (Transonic Systems, Ithaca, NY) for measurement of cardiac output and uterine blood flow. These surgical procedures have been described in detail previously (2). Briefly, nonpregnant ewes were fasted and water was withheld for 24 h prior to surgery. The ewes were sedated with pentobarbital sodium (15 mg/kg), intubated endotracheally, connected to a veterinary anesthesia machine, and ventilated with a mixture of 2-3% isoflurane and oxygen. An incision was made through the left, fourth intercostal space, and the pulmonary artery was isolated and fitted with a 24-mm Transonic flow probe for determination of cardiac output and calculation of systemic vascular resistance. A chest tube, connected to a hemovac, was inserted during the thoracotomy and remained in the ewe for 72 h following surgery to drain the chest cavity of excess fluids and air. The flow probe cable was exteriorized via a subcutaneous tunnel and placed in a cloth pouch on the animal's left shoulder. After the procedure, ewes were placed in portable cages and received water and food ad libitum.

After a 1-wk recovery period, the ewes were again fasted and water was withheld for 24 h prior to the laparotomy. Ewes received pentobarbital sodium sedation and isoflurane anesthesia as described previously. An incision was made in the left groin over the femoral vessels. The femoral artery and vein were cannulated with polyvinyl catheters to the level of the distal aorta and vena cava. A midline, vertical abdominal incision was made to expose the uterus. Both uterine arteries were dissected free and fitted with 2- or 3-mm Transonic Doppler flow probes for continuous uterine blood flow determinations. A lateral branch of each uterine artery was cannulated to infuse the drugs under study directly into the uterine vasculature. A bilateral oophorectomy was then performed to control exposure of the ewe to circulating sex steroids. The abdominal and groin incisions were closed in layers, and the catheters and flow probes were exteriorized via a subcutaneous tunnel and placed in a cloth pouch on the left flank.

Animals were allowed a minimum of 5 days to recover from the second surgery. Ewes received 1 µg/kg of 17-estradiol intravenously daily, and all catheters were flushed with heparin solution to maintain patency. The experimental protocols were spaced at least 72 h apart to allow for total clearance of study drugs. All surgical procedures were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.

Drug preparations. The AT₁-receptor antagonist L-158809 was provided by Merck (West Point, PA), whereas the AT₂- receptor antagonist PD-123319 was provided by Parke-Davis (Ann Arbor, MI). Both drugs were prepared according to literature provided by the pharmaceutical companies. L-158809 was diluted in phosphate-buffered saline at pH 8.6 to a final concentration of 10 mg/ml. PD-123319 was diluted in phosphate-buffered saline at pH 3.2 to a final concentration of 10 mg/ml. At the doses used in these studies, both of these peptide antagonists have been shown to be specific for their subtype receptor and to produce only antagonist properties by direct competitive inhibition (6, 11, 18). ANG II (Sigma Chemical, St. Louis, MO) was prepared with normal saline and stored frozen in 10 µg/ml aliquots, which were thawed for each experiment. Appropriate pH-matched vehicles were also evaluated.

Experimental protocol 1. The purpose of the first experimental protocol was to determine whether ANG II is a direct vasoconstrictor of the ovine uterine vasculature and to evaluate which ANG II receptor subtype (1 or 2) mediates uterine and systemic vasoconstriction in the nonpregnant sheep. Ewes were allowed to acclimate for 30 min prior to receiving 1 µg/kg of 17-estradiol intravenously. Within 2 h after the estradiol administration, uterine blood flow increased from a baseline of 12 ± 3 to 152 ± 32 ml/min. Once uterine blood flow had increased, it remained stable for approximately 90 min. It was during this steady state of maximal uterine blood flow that the experimental protocol was performed. ANG II was administered into the uterine artery as a series of bolus injections of increasing dose (0.1, 0.3, 1.0 µg) to determine control uterine and systemic vascular responses. Each bolus was administered over a 20-s period. Measurements were obtained at the peak of the response to ANG II (within 60 s of the bolus).

Experimental protocol 2. To ascertain whether similar uterine responses occur when ANG II is given systemically, ANG II was infused in separate experiments into the femoral vein at the rate of 100 ng · kg¹ · min¹ for 10 min. Once control responses to ANG II were obtained, PD-123319 (AT₂ blockade) was given (3 mg/min for 5 min). The response to systemic ANG II was reevaluated at its peak response. The effects of AT₁-receptor blockade during the systemic ANG II infusion was determined by infusing L-158809 at 0.3 mg/min for 5 min and then repeating the systemic ANG II infusion. Once this was complete, we increased the AT₁-receptor blockade dose to 3 mg/min for 5 min (total of 15 mg) and once again repeated the systemic infusion.

Experimental protocol 3. This protocol was designed to investigate the mechanism by which ANG II uterine vascular responses were potentiated in the presence of AT₂ blockade (see RESULTS and Fig. 2, A and B). We sought to selectively stimulate the AT₂ receptor in the presence of AT₁-receptor blockade using L-158809. In these studies, ewes did not receive estrogen prior to this protocol to allow a vasodilatory response to be detected. An intrauterine artery infusion of ANG II was given for 10 min at a rate of 0.03 µg/min, which has previously been shown to reduce uterine blood flow by approximately 50%. After the uterine blood flow returned to baseline, the AT₁ antagonist L-158809 (3 mg/min for 5 min) was infused into the uterine artery. The infusion of ANG II was then repeated, and increases in uterine blood flow from baseline were observed. To determine whether this vasodilation was mediated by AT₂ receptors, PD-123319 (3 mg/min for 5 min) was infused into the uterine artery and then the ANG II infusion was repeated in four ewes. Finally, to determine whether the vasodilation was mediated by nitric oxide or a prostaglandin such as prostacyclin, animals received either N-nitro-L-arginine methyl ester (L-NAME) as a 10-mg bolus into the uterine artery or indomethacin (2 mg/kg) in the femoral vein. Uterine responses were then determined. Sixty minutes were allowed to elapse after the indomethacin was given.

Statistical analysis. Data are reported as means ± SE, either actual or percent change from baseline response. Multiple comparisons were determined by a two-way ANOVA or Student's paired t-test as appropriate. Comparisons within and between groups were made with ANOVA, and mean differences were determined with the appropriate post hoc test. P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ANG II as a direct uterine vasoconstrictor. In representative tracings, the mean arterial pressure (Fig. 1A) and uterine blood flows in the experimental and control (contralateral) sides (Fig. 1, B and C) are shown. A significant decrease in uterine blood flow is seen with an intrauterine arterial bolus injection of 1.0 µg of ANG II. This decrease in uterine blood flow is seen prior to any changes noticed in the mean arterial pressure. There are no changes in uterine blood flow in the contralateral uterine artery. This fact supports our hypothesis that ANG II acts as a direct vasoconstrictor in the uterine vasculature of the nonpregnant sheep and that the vasoconstriction seen with its infusion is due to local effects of ANG II and does not reflect changes in the systemic vasculature.

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Representative tracing of mean arterial pressure (MAP, A) and uterine blood flow (UBF) during 1.0-µg bolus of angiotensin II (ANG II), indicated by arrow, in experimental uterine artery (B). Contralateral uterine blood flow is shown in C.

Experimental protocol 1: Intrauterine artery administration of ANG II. ANG II produced dose-related decreases in ipsilateral uterine blood flow in estrogenized ewes. Figure 2A illustrates the percent decrease in uterine blood flow from the estrogenized baseline (152 ± 8 ml/min) in response to bolus injections of 0.1, 0.3 and 1.0 µg of ANG II. Pretreatment of animals with the AT₂- receptor antagonist PD-1233l9 significantly potentiated (P < 0.02) the ANG II vasoconstrictor responses at the two highest doses. Subsequent treatment of the uterine vasculature with the AT₁-receptor antagonist L-158809 at 0.3 mg/min significantly inhibited the vasoconstriction seen with ANG II and the 3.0 mg/min dose abolished the vasoconstriction in the uterine vasculature (P < 0.01).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Intrauterine administration of ANG II. A: percent decreases in uterine blood flow from baseline in response to intrauterine arterial bolus injections of ANG II, either alone or in the presence of ANG II type 1 and 2 (AT1 or AT2)-receptor blockade. * P < 0.05 vs. controls. B: percent increases in calculated uterine vascular resistance from baseline in response to intrauterine arterial bolus injections of ANG II either alone or in presence of AT1- or AT2-receptor blockade. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. controls. C: percent increase in mean arterial pressure from baseline in response to intrauterine arterial bolus injections of ANG II, either alone or in presence of AT1- or AT2-receptor blockade. ** P < 0.01, *** P < 0.001 vs. controls.

Experimental protocol 2: Systemic administration of ANG II. Figure 3 shows the time relationship of mean arterial pressure, cardiac output, uterine blood flow, and calculated systemic and uterine vascular resistance during a 5-min infusion of ANG II (100 ng · kg¹ · min¹). During the system infusion of ANG II, systemic vascular resistance increased, first reaching a peak at 2 min, and this is followed by a peak in uterine vascular resistance at 4 min. Mean arterial pressure reaches a peak at approximately 1 min, but uterine blood flow remains unchanged initially because the increased perfusion pressure is able to overcome the increase in uterine vascular resistance. The rise in uterine and systemic vascular resistance occurs almost simultaneously.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Percent change from baseline over time in response to systemic ANG II administration of 100 ng · kg1 · min1. Cardiac output (CO), urine blood flow (UBF), uterine vascular resistance (UVR), systemic vascular resistance (SVR), and mean arterial pressure (MAP).

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View larger version (27K): [in this window] [in a new window]   Fig. 4.   Systemic administration of ANG II. A: percent increases in mean arterial pressure from baseline in response to systemic infusion of ANG II either alone or in presence of AT1- or AT2-receptor blockade. ** P < 0.01, *** P < 0.001 vs. controls. B: percent increases in calculated systemic vascular resistance from baseline in response to systemic infusion of ANG II either alone or in presence of AT1- or AT2-receptor blockade. *** P < 0.001 vs. controls. C: percent decreases in uterine blood flow from baseline in response to systemic infusion of ANG II either alone or in presence of AT1- or AT2-receptor blockade. * P < 0.05 vs. controls. D: percent increases in calculated uterine vascular resistance from baseline in response to systemic infusion of ANG II either alone or in presence of AT1- or AT2-receptor blockade. ** P < 0.01, *** P < 0.001 vs. controls.

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Experimental protocol 3. Figure 5 illustrates the uterine blood flow changes from baseline in response in nonestrogenized ewes to local intrauterine arterial administration of angiotensin of 0.03 µg/min before and after AT₁-receptor blockade. ANG II produced a significant decrease in uterine blood flow by 64 ± 3%, from a baseline of 17 ± 4 ml/min. After administration of the AT₁ antagonist into the uterine vasculature, a significant vasodilatory response was seen with ANG II infusion. To determine whether the vasodilation was due to AT₂ stimulation, responses were determined following AT₂-receptor blockade. As shown in Fig. 6, the vasodilatory response was reversed when the ANG II type 2 receptor was blocked (P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of intrauterine artery ANG II infusion on uterine blood flow changes before and after AT1 antagonist. * P < 0.05 vs. baseline.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of intrauterine artery ANG II infusion plus AT1 blockade on uterine blood flow before and after AT2 blockade. * P < 0.05 vs. baseline.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effect of intrauterine artery ANG II infusion plus AT1-receptor blockade on uterine blood flow before and after N-nitro-L-arginine methyl ester (L-NAME). * P < 0.05 vs. baseline.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our hypothesis for the present research was that uterine vasoconstriction in response to ANG II is mediated by AT₂ receptors because this is the predominant vascular receptor subtype in the pregnant and nonpregnant ovine uterus (3). However, in contrast to our initial hypothesis, the data from the present study indicate that in the ewe ANG II-stimulated uterine vasoconstriction is mediated by the AT₁ receptors. When these receptors were selectively antagonized, both the uterine and systemic vasoconstriction seen with ANG II was completely ablated. This was independent of the route of ANG II administration, local or systemic. These data are consistent with those of other studies, which have demonstrated that in tissues where AT₁ and AT₂ receptors coexist, vasoconstriction that occurs with ANG II is mediated entirely by the AT₁ subtype (6, 18, 21).

Although a definite function of the AT₂ receptor was not determined in the present study, a significant potentiation of the ANG II-induced decreases in uterine blood flow and increases in uterine vascular resistance was observed following pretreatment with PD-123319, the AT₂-receptor antagonist. This potentiation within the uterine vasculature was observed following both local and systemic ANG II administration. To our knowledge, this is the first time that potentiation of the vasoconstrictive effects of ANG II have been demonstrated with the inhibition of the AT₂ receptor. Two possible explanations exist. First, stimulation of AT₂ receptors could produce local endothelial or vascular smooth muscle production of a vasodilator such as nitric oxide or prostacyclin, which in turn could antagonize the vasoconstrictor responses to ANG II, mediated via the AT₁ receptor. One of the novel observations that we made in the present study suggests that nitric oxide is released when ANG II is administered in the presence of AT₁-receptor blockade. Second, PD-123319 could have some AT₁-receptor agonistic properties such that the combination of ANG II and PD-123319 would lead to greater vascular responses. This, however, appears unlikely because administration of PD-123319 directly into the uterine artery at the doses used in the present study had no observable vasoactive properties. The first possibility seems the most logical based on our data as well as on several reports in the literature (8, 9) that describe an increased vasopressor response to ANG II in mice lacking the AT₂ receptor. Also, Magness and co-workers (13) have shown that ANG II stimulates nitric oxide production in the uterine arteries of pregnant and nonpregnant ewes.

Although AT₂-receptor blockade potentiated uterine vasoconstrictor responses to ANG II, systemic responses did not show similar modulation. In fact, in contrast to the potentiation of ANG II responses observed in the uterine vasculature following uterine artery PD-123319 administration, systemic responses were actually significantly reduced (Fig. 2C). Thus, although the dose of PD-123319 was high enough to have an effect on systemic arterial pressure responses to ANG II, no potentiation was observed. The mechanism by which this occurs is presently unknown.

The ANG II antagonists used in this study, L-158809 and PD-123319, are known to be specific for their respective receptors, AT₁ and AT₂, in vitro at concentrations of less than 10⁵ M (4, 10, 17). The in vivo concentrations of the antagonists can be estimated from our infusion. By our calculations, the infusion of the antagonists at 3 mg/min for 5 min with an average uterine blood flow of 152 ml/min is approximately 4 × 10⁵ M at the peak. Systemic doses of the antagonists L-158809 and PD-123319 produced similar responses. Local infusions of the antagonists were selected in an attempt to ensure that the receptors in the uterus, and not just the systemic receptors, would be bound.

The renin-angiotensin system has been extensively studied in both the pregnant and the nonpregnant human as well as in animal models. In normal human pregnancy, serum levels of the components of the renin-angiotensin system (including renin, angiotensin, and aldosterone) are elevated compared with the nonpregnant state (16, 20). Yet, normal pregnant women are resistant to the vasoconstrictor effects of infused ANG II (7). This refractoriness may be very important because uterine blood flow steadily increases from 2% of cardiac output in the nonpregnant state up to 20% of cardiac output in late pregnancy, reaching a maximum value of 500 ml/min at term in humans (19) and 1,200 ml/min in the ewe (17). Additionally, it is clear that diseases such as preeclampsia, which are associated with increased sensitivity to ANG II, are also associated with reduced uterine blood flow (1). Circulating ANG II levels in the mother and fetus, which are elevated in pregnancy, appear to be important in the regulation of uterine and/or umbilical blood flow because the use of ACE inhibitors in pregnancy is associated with a decrease in placental blood flow and oxygen delivery in the pregnant ewe (12, 15). ACE inhibitors are contraindicated in pregnancy because fetal hypoxia, fetal growth restriction, oligohydramnios, renal dysplasia, and intrauterine fetal death are noted with their use (10a). The fetal effects seen with ACE inhibitors are in part associated with the changes in uteroplacental blood flow and subsequent decreased perfusion of the fetus. Thus it appears that ANG II plays a role in the regulation of uteroplacental and umbilical blood flow dynamics, because inhibition of its actions is deleterious to the fetus. The present study supports the concept that ANG II may be an important regulator of uterine blood flow in the ewe, inasmuch as blockage of both AT₁ and AT₂ receptors modulates the vascular responses to ANG II in the uterus.

In the present study, we have evaluated whether ANG II is a direct-acting vasoconstrictor or acts by releasing a systemic vasoconstrictor as has been suggested by others (4). In the present study, it is clear that when ANG II is administered as an intrauterine artery bolus local vasoconstriction occurs because the vasoconstriction is only seen in the uterine horn, which receives the ANG II, and not in the contralateral horn. Furthermore, systemic infusion of ANG II, which increases systemic vascular resistance, appears to increase uterine vascular resistance. Within 30 s of beginning the ANG II infusion, mean arterial pressure is increased by 9%, systemic vascular resistance has increased by 43%, and uterine vascular resistance is increased by 28%, whereas uterine blood flow is not changed. Thus, almost immediately, uterine vascular resistance begins to rise, indicating that vasoconstriction is occurring.

In conclusion, although the predominant ANG II receptor in both affinity and number in the ovine uterine vasculature is the AT₂ subtype, the vasoconstriction produced by ANG II appears to be mediated by the AT₁ receptor. Furthermore, blockade of the AT₂ receptor resulted in a potentiation of the uterine vasoconstrictor response to angiotensin, suggesting that the AT₂ receptor may serve a vital role in modulating uterine blood flow responses to ANG II. Although possible, it is not currently clear whether selective stimulation of AT₂ receptors will result in direct uterine vasodilation. If this mechanism occurs in pregnancy then this may in part explain why ACE inhibitors, which block ANG II formation, could cause reduced uteroplacental blood flow.