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The RAS has been considered an important modulator of cardiovascular changes during the perinatal period (1–4). ANG II, the predominant vasoactive agent associated with the RAS, is believed to contribute to the regulation of basal and stress-induced changes in blood flow and blood pressure in fetal, neonatal, and adult sheep (3–6). It mediates its actions by binding to specific ANG II receptors (ATRs). In adults, including sheep, the AT1 receptor subtype (AT1R) is the primary receptor expressed in most tissues and mediates the majority of biologic responses to ANG II, including its effects on VSM and arterial pressure (7,8). However, AT2R, which does not mediate vasoconstriction, is the predominant receptor in fetal tissues, including the peripheral VSM, and persists in the postnatal period (7,9–11). In newborn sheep, the transition from the AT2R to AT1R in VSM begins 2–4 wk after birth and is not complete until 3 mo (9,10). Although the definitive roles of the VSM AT2R during development are unclear (7), it does not mediate vasoconstrictor responses, but may contribute to the regulation of VSM maturation (12–14) and/or the attenuation of AT1R-mediated responses by the VSM (7,15,16).

Basal MAP increases during the first month of life in neonates of several species (17–20), but vascular responses to systemic ANG II infusions are unchanged (17,21). Unlike an α-agonist or arginine vasopressin (AVP), arterial ANG II infusions in the hind limb of conscious newborn sheep do not elicit changes in blood flow or peripheral vascular resistance (PVR), suggesting that it does not directly alter PVR (17,18). Therefore, the absence of an increase in vascular reactivity or sensitivity to infused ANG II with increasing postnatal age could be due to persistent AT2R expression in peripheral VSM during the first postnatal month (9,10) or the stage of VSM functional maturation (22). Alternatively, the MCRANG II may increase with increasing postnatal age, resulting in an apparent attenuation of the responses to systemic ANG II infusions. For example, the pressor responses to comparable doses of infused ANG II by fetal sheep were reported to be attenuated compared with those of adult animals (23). However, we (24) subsequently reported that these attenuated fetal pressor responses were due to a fetal MCRANG II that was more than ninefold higher than the adult. Thus, when pressor responses were examined at comparable plasma levels of ANG II, there was no difference in fetal and maternal responses to infused ANG II. It is unclear whether the absence of an increase in the pressor responsiveness to infused ANG II in the first postnatal month is also due to alterations in MCRANG II. Furthermore, the activity of the RAS has not been well characterized in the newborn of any species. The present study was designed to examine and characterize the relationships between basal MAP, arterial levels of ANG II and PRA, and the MCRANGII in conscious, chronically instrumented newborn sheep during the first 5 wk postnatally.

MATERIALS AND METHODS

Animal preparation.

Nine newborn singleton sheep (five males, four females) were studied between 5 and 35 d postnatally. The animal preparation has been described (17,18). Briefly, after delivery at our facility, surgery was performed at 2–5 d of life. Preoperatively, animals received subcutaneous (s.c.) atropine (0.088 mg/kg) followed by intravenous (i.v.) sodium pentobarbital (7.5–10 mg/kg) and 1% ketamine hydrochloride (1–2 mg/kg) via a percutaneous jugular venous catheter. During surgery, 1% ketamine hydrochloride (1 mg/kg i.v.) was given to maintain deep anesthesia. Through an inguinal incision in one hind limb, the femoral artery and vein were identified, dissected, and two polyvinyl catheters containing heparinized saline (250 U/mL) were implanted at 12.5 and 7.5 cm in the femoral artery for cardiovascular monitoring and blood sampling. A femoral venous catheter was inserted at 15 cm, placing the tip above the bifurcation of the inferior vena cava, and used for systemic ANG II infusions. The catheters were flushed with heparinized saline (250 U/mL), closed with sterile metal pins, brought out to the flank through an s.c. tunnel, and placed in a canvas pouch attached to the skin with sterile steel pins. The skin incisions were closed with surgical staples. During surgery, animals received 10% dextrose and isotonic saline at 20 mL/kg·h to maintain plasma glucose and volume. All animals received i.v. banamine (0.1 mL; Schering-Plough Animal Health, Union, NJ) for pain and intramuscular penicillin (60,000 U) and gentamicin (7.5 mg) for prophylaxis after surgery and the next 2 d postoperatively. After surgery, animals received 70 mL/kg of 5% dextrose and isotonic saline over 4 h and were maintained under a heated warmer until they recovered from anesthesia. They were returned to their mothers for feeding, but were removed the first night and kept in a recovery facility in the Animal Resources Center for monitoring and bottle feeding. Subsequently, each animal was kept with its mother except during an experiment. Catheters were flushed daily with heparinized saline (250 U/mL) to maintain patency. All animals received prophylactic intramuscular penicillin (60,000 U) and gentamicin (7.5 mg) at the completion of each study. These studies were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center.

Experimental protocol.

Studies were performed after the animals were considered to have recovered from surgery and were eating; no animal was studied before the second postoperative day. Studies were performed in a sling after a 40-min control period and demonstrating stable hemodynamic parameters and arterial blood gases (17,18). Two doses of ANG II (angiotensin amide, Sigma Chemical Co., St. Louis, MO), 0.573 and 1.15 μg/min that had previously been studied in our laboratory were selected for these studies (11,23). However, because each animal was growing and would weigh differently at the time of each study, doses were changed to 0.076 and 0.19 μg/kg·min to account for expected growth and change in the volume of distribution. The use of two doses permitted us to determine whether the MCRANG II was dose related. After the stabilization period, ANG II was infused via the femoral venous catheter using a constant infusion pump (Harvard Apparatus, S. Natick, MA). The order of doses was randomly chosen, and infusions were maintained for 20 min while MAP and heart rate were continuously monitored. Blood samples (3 mL) for measurements of plasma ANG II and/or PRA were obtained from the femoral arterial catheter after stabilization, before beginning the ANG II infusion (control) and at 5, 10, 15, and 20 min during the 20-min infusion. MAP and heart rate were continuously monitored with pressure transducers (P23XL, Gould, Cleveland, OH), and the signals recorded on an eight-channel recorder (model RS 3800, Gould Corp.) and PONEMAH Data Acquisition System (Gould Corp.).

ANG II assay.

When the femoral arterial catheter was patent, blood samples (3 mL) were collected as described above. Additional arterial samples were taken from an adult nonpregnant ewe to assess recovery and compare with previous levels from our laboratories (25). Blood samples were collected in chilled syringes, placed in chilled tubes containing ethylenediamine tetraacetic acid and 1,10-phenanthroline (monohydrate) (Sigma Chemical Co.-Aldrich, St. Louis, MO), centrifuged at 1200 rpm at 4°C for 15 min, and the plasma removed and placed in 75 × 12-mm polystyrene tubes. The plasma was stored at −20°C. To determine recovery, one set of maternal plasma samples was prepared unmodified, and a second and third set had 200 or 1000 pg/mL of ANG II added before freezing. At the time of assay, plasma samples were thawed and the extraction procedure outlined in the enzyme immunoassay kit (Peninsula Laboratories, Inc., San Carlos, CA) was used; however, we substituted the C18 columns with 100 mg phenyl columns (Varian, Harbor City, CA). After extraction, the eluant was collected in 1.5-mL microcentrifuge tubes, placed in an integrated Speedvac system ISS110 (Savant Instruments, Inc., Farmingdale, NY) to evaporate to dryness, and reconstituted using assay buffer. Samples were read in a microplate reader (Bio-tek Instruments, Inc., Winooski, VT) at 450 nm. The intra-assay and interassay variability were 3.59% and 11%, respectively. Recovery was >80%.

MCRANG II.

The MCRANG II was calculated as previously described (24–27). This assumes that the endogenous production rate of ANG II is small compared with the rate of ANG II infusion and a steady state has been achieved. When this occurs, the MCR is calculated with the following equation: MCRANG II = I/Cp · W, where MCRANG II is ANG II clearance in mL/min·kg body weight, Cp is the ANG II plasma concentration in the steady state in pg/mL, I is the rate of ANG II infusion in ng/min, and W is the weight in kg at each study. MCRANG II was calculated at 15 and 20 min of infusion.

PRA.

PRA was measured as ANG I generation with a commercial kit (RIANEN, New England Nuclear, North Billerica, MA) and is expressed as nanograms of ANG I formed per milliliter of plasma per hour. The use and accuracy of this assay in our laboratories have been reported (28).

Statistical analyses.

Linear and nonlinear regression analyses were used to examine changes over time or with a dose of ANG II. Analysis of variance (ANOVA) for multiple groups was used to compare data grouped by postnatal age followed by the appropriate post hoc test. In this instance, studies were grouped as <10 d, 10–20 d, and 21–35 d postnatally; however, the n values presented will differ based on the data analyzed. For example, although there were 17 studies of MCR in nine animals, two doses were examined in four experiments on the same day and baseline data cannot be counted twice. Therefore, in four experiments, the preinfusion values were averaged, resulting in 13 values for basal ANG II, MAP, and heart rate. Also note that two samples were not available for PRA measurements. p Values are presented for each analysis. Data are presented as the means ± SEM.

RESULTS

Baseline parameters.

Basal MAP increased during the postnatal study period (Table 1; p = 0.015 by ANOVA), increasing 14% at 1 mo, whereas heart rate decreased 24% (p = 0.06). Arterial blood gases obtained before infusion of each ANG II dose at each age were within normal ranges for our laboratory and similar between age groups. Although MAP increased during the first month, basal arterial levels of ANG II progressively decreased (Fig. 1), decreasing from 942 ± 230 pg/mL (n = 3) at <10 d postnatally to 471 ± 152 (n = 6) and 240 ± 70 (n = 4) pg/mL at 10–20 d and 21–35 d (p = 0.05, ANOVA), respectively. In contrast, basal PRA was increased and unchanged throughout the first postnatal month (r = 0.03, p = 0.92), averaging 6.70 ± 1.1 ng/mL·h.

Table 1 Physiologic parameters before the systemic infusion of ANG II in conscious newborn sheep
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Figure 1
figure 1

Baseline levels of arterial angiotensin II in conscious newborn sheep during the first postnatal month. Values are grouped by age: <10 d (n = 3), 10–20 d (n = 6), and >20 d (n = 4). p = 0.05, ANOVA.

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Cardiovascular responses to infused ANG II.

At each age, the measurements of basal MAP and heart rate did not differ before infusion of either dose of ANG II (Table 2). As anticipated (17), systemic infusions of ANG II increased MAP dose dependently (p = 0.019) at each age. Steady-state pressor responses were achieved within 2 min and remained unchanged throughout the 20-min infusion (p > 0.1, ANOVA). There was no increase in the relative MAP responses, i.e. the percent rise, with increasing postnatal age, demonstrating that the responsiveness to ANG II was unchanged. Heart rate decreased during ANG II infusions, and similar to MAP, achieved a steady-state response that was unchanged during the continuous infusions.

Table 2 MAP, heart rate, and arterial plasma levels of ANG II in conscious newborn sheep before and during the systemic infusions of ANG II in the first month of life
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ANG II metabolic clearance.

To measure the MCR of any substance, the rate of infusion should exceed the endogenous production rate, and a steady-state plasma level of the substance must be achieved (24–27). Arterial levels of ANG II achieved steady-state levels within 5 min (data not shown) of initiating the systemic ANG II infusion with each dose and at each age studied (p = 0.007, ANOVA), except for 0.191 μg/min·kg at <10 d, and a steady-state arterial concentration was maintained at 15 and 20 min during all studies (Table 2). When we compared the MCRANG II obtained with the two primary doses studied, values were 80% higher with 0.191 versus 0.076 μg/min·kg, 145 ± 23 (n = 6) versus 81.3 ± 7.1 (n = 8) mL/min·kg (p = 0.01). This dose-dependent response in MCRANG II was also observed when measurements at other rates of infusion were included in the analysis (Fig. 2; r = 0.95, p < 0.001, n = 17). There was, however, no relationship between postnatal age and MCRANG II (Fig. 3; r = 0.32, p = 0.2, n = 17) with either rate of infusion. There also was no relationship between basal ANG II levels and MCRANG II (data not shown, p > 0.1).

Figure 2
figure 2

Relationship between MCR ANG II and rate of ANG II infusion in conscious newborn sheep (n = 17).

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Figure 3
figure 3

Relationship between MCRANG II and postnatal age in conscious newborn sheep. Open circles represent MCRANG II at infusion rate of 0.076 μg/kg·min (n = 8), filled circles at 0.191 μg/kg·min (n = 6) and filled triangles with other doses (n = 3).

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Because the arterial levels of PRA remained increased throughout the study period, despite increased levels of circulating ANG II, it was unclear whether the negative feedback mechanism of ANG II on PRA was intact. Therefore, we examined simultaneous concentrations of ANG II and PRA at 15 and 20 min of infusion. At both times, the increase in ANG II was associated with a reciprocal decrease in PRA, e.g. at 15 min ANG II increased 160%, and PRA decreased 35% from 6.93 ± 1.2 to 4.47 ± 0.86 (Fig. 4, p < 0.01).

Figure 4
figure 4

Relationship between arterial plasma ANG II (A) and PRA (B) before and at 15 min of ANG II infusion in conscious newborn sheep (n = 10). Horizontal lines represent means. *p ≤ 0.004.

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DISCUSSION

During the transition from the fetal to neonatal state, there is an increase in MAP associated with removal of the low resistance placental vascular bed and increased circulating levels of ANG II, catecholamines, and AVP (1,29–31). Basal MAP increases further during the next 2–4 wk (17–20). The contributions of the RAS to the increase in MAP at birth and subsequent weeks are unclear. In term newborn sheep, the AT2R, which does not mediate vasoconstriction, persists in peripheral VSM until >4 wk postnatally (9). Although infused ANG II increases MAP, vascular sensitivity is unchanged and PVR is unaffected (17,21). The constant vascular sensitivity could reflect an increased MCRANG II in the newborn, which has not been studied, resulting in the rapid removal of infused ANG II and an apparent attenuation of vascular responsiveness as in fetal sheep (24). In the present study, we examined the activity of the RAS and ANG II metabolism in conscious sheep in the first postnatal month. We observed 1) increased basal circulating levels of ANG II compared with the adult and a gradual decrease as MAP increased, 2) stable vascular responsiveness to infused ANG II after birth that does not reflect changes in MCRANG II, and 3) persistent increase in PRA after birth, suggesting enhancement of RAS activity. Thus, MCRANG II does not explain the stable responses to infused ANG II, and persistent postnatal AT2R expression in peripheral VSM may protect the newborn from the hypertensive effects of excessive ANG II synthesis after birth (9,10).

The increase in basal MAP immediately after birth is largely due to removal of the low resistance placental circulation, which accounts for 40% of cardiac output (32) and maintenance of a high cardiac output (33). In fetal sheep, the RAS was thought to regulate MAP by increasing PVR (4). However, Kaiser et al. (11) reported that ANG II did not alter PVR in term fetal sheep, but rather contributed to MAP primarily by increasing umbilicoplacental vascular resistance. This reflected the advanced maturation and expression of AT1R in umbilical artery VSM in contrast to peripheral VSM, which was functionally less mature and expressed AT2R (9,10,22). The AT2R is persistently expressed in peripheral VSM of newborn sheep (9,10). Moreover, as in fetal sheep (11), arterial ANG II infusions in the newborn hind limb do not elicit vasoconstrictor responses (17), suggesting that ANG II–induced pressor responses after birth occur through indirect mechanisms, e.g. release of AVP or catecholamines (34,35). However, Wilson et al. (21) reported that increases in circulating ANG II after birth contributed to the increase in MAP. In contrast, we observed that circulating ANG II decreased in the first postnatal month and was inversely related to basal MAP. Our findings are strikingly similar to those of Broughton et al. (3), who also observed increased circulating ANG II–like activity after birth and a decrease from 839 ± 96 pg/mL within 8 h to 315 ± 49 pg/mL between 10 h and 7 d and <111 ± 15 pg/mL at 6–8 wk. We conclude that plasma ANG II is inversely related to the increase in postnatal MAP, making it unclear how ANG II contributes to postnatal blood pressure regulation.

In adult VSM, ANG II mediates its pressor effects by binding to AT1R (7,8,16). If AT1R predominated in VSM after birth, a marked increase in systemic MAP would have been expected followed by persistent hypertension because of the higher levels of circulating ANG II observed by us and Broughton et al. (3). This does not occur, suggesting an immature receptor signaling mechanism in newborn VSM, incomplete biochemical development of VSM contractile function (22,36), or enhanced vascular synthesis of an ANG II antagonist, e.g. prostacycline or nitric oxide (37). Alternatively, persistent expression of AT2R, which does not elicit contraction responses, in neonatal VSM could explain why MAP was not higher. This is supported by observations that AT1R binding in newborn VSM does not occur until >2 wk postnatally, and a complete switch to AT1R occurs after 3 mo postnatally (9). The increase in AT1R binding (9) and expression (10), however, parallels the increase in postnatal ovine MAP (17,18). Because this is associated with decreased circulating ANG II, it is intriguing to consider that the decrease in plasma ANG II contributes to the increase in AT1R expression or receptor avidity in newborn VSM (38); this has not been studied. Although plasma ANG II decreased 75% in the first month, levels remained fourfold greater than adult values, 40 pg/mL (24,25,27). Thus, it remains unclear why basal MAP is not higher.

Before birth, MCRANG II is 700 mL/min·kg (24,39), PRA is >10 ng/mL·h (Fig. 5), and plasma ANG II is 40–70 pg/mL, demonstrating enhanced activity of the fetal RAS and high ANG II production. The placental vascular bed accounts for 85%–90% of fetal ANG II clearance (24). Thus, the marked elevation in circulating ANG II immediately after birth (1,3) reflects removal of the placenta and persistence of the high ANG II synthesis necessary to maintain fetal plasma ANG II. If placental clearance accounts of >85% of fetal MCRANG II, nonplacental clearance is 100 mL/min·kg. Notably, MCRANG II was 115 mL/min·kg postnatally, resembling values in adult women and sheep (25,27). However, unlike the fetus and adult, the MCRANG II increased with the higher ANG II dose, suggesting that this may be a mechanism for regulating circulating ANG II levels. Thus, we demonstrate for the first time that nonplacental ANG II metabolism is similar in the developing animal and adult, increased plasma ANG II at birth reflects an increased production rate consistent with PRA values three times greater than adult sheep (Fig. 5), and MCRANG II in the neonate is concentration dependent. We also provide evidence that the MCRANG II is not responsible for the stable vascular responses to infused ANG II in newborn sheep (17,21). Therefore, other mechanisms, not yet determined, are involved (37,40).

Figure 5
figure 5

Comparison of arterial PRA in fetal (n = 5), newborn (n = 15), nonpregnant (n = 13), and pregnant (n = 6) sheep. Different symbols represent group differences at p < 0.001, ANOVA. Values for fetal and adult sheep extracted from Kaiser et al. (11) and Matsuura et al. (28), respectively.

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ANG II is synthesized from ANG I, which is liberated by the action of renin on angiotensinogen. ANG II normally feeds back to inhibit renin secretion by a direct action on juxtaglomerular cells in the kidney via AT1R, regulating circulating ANG II by a negative feedback mechanism that modifies ANG I levels. Thus, an inverse correlation between plasma ANG II and PRA normally exists (41,42). In this study, basal PRA remained increased, although plasma ANG II was markedly increased. Thus, the negative feedback mechanism was not intact or the ANG II concentration required for activation was up-regulated (43–45). As in fetal and adult sheep (11,24,25), acute elevations in ANG II decreased PRA, demonstrating an intact feedback mechanism. The persistent increase in PRA, despite increased ANG II levels, suggests RAS up-regulation before and after birth, resulting in an increased set point and attenuated feedback mechanism. This is consistent with previous observations of decreased pressor responses to infused ANG II when PRA is increased (46). Although PRA decreases with increasing age (Fig. 5), this occurs after the first postnatal month. Further, it is unclear why postnatal PRA remains increased and whether angiotensinogen or renin is also increased.

In this study, increased circulating ANG II and PRA persisted in the first postnatal month, demonstrating increased RAS activity. There also was an 83% decrease in MCRANG II after birth, achieving values resembling fetal and adult nonplacental clearance. Although PRA was increased and appeared unresponsive to increased circulating ANG II, the negative feedback mechanism was intact, suggesting up-regulation of the set point, which allows enhanced ANG II synthesis, but the purpose is unclear. We suggest for the first time that persistent AT2R expression in newborn VSM (9,10) prevents the hypertensive effects of increased ANG II and may also attenuate vasoconstrictor effects by emerging AT1R (15). These data provide new insights into the RAS during development and its role in blood pressure regulation. Further studies are needed to clarify the role of the RAS during development.