INTRODUCTION

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NITRIC OXIDE (NO) is a labile compound produced via the oxidation of a guanidino-nitrogen moiety on the amino acid L-arginine, a reaction catalyzed by the ubiquitous enzyme NO synthase (26). Within the peripheral vasculature, NO is constitutively produced within endothelial cells and plays an important role in regulating vascular resistance by stimulating soluble guanylate cyclase in subjacent smooth muscle cells, with consequent elevation of cGMP to produce vasorelaxation (26). Moreover, inhibition of NO synthase with L-arginine analogs in adult experimental animals is accompanied by a dose-dependent rise in blood pressure and fall in cardiac output, consistent with the notion that NO maintains a tonic vasodilatory influence within the circulation (26, 34). However, despite the fall in cardiac output and consequent reduction in systemic O₂ delivery, inhibition of NO synthase is accompanied by a proportionally greater increase in tissue O₂ extraction, so that whole body O₂ consumption rises (34). In conjunction with in vitro observations, which indicate that NO inhibits mitochondrial respiratory processes (39), the latter provides evidence that NO normally exerts a suppressive effect on tissue oxidative metabolism in the adult.

As in the adult, inhibition of NO synthesis increases fetal arterial blood pressure (5, 11, 27) and reduces fetal cardiac output (11), but it is unknown if NO additionally modulates fetal whole body O₂ extraction or O₂ consumption. The latter question is of particular relevance because of the major differences that exist between the adult and fetal circulations. Thus the low fetal blood O₂ content not only requires a high level of cardiac output to maintain an adequate level of whole body O₂ delivery (31) but may also limit any increases in systemic O₂ extraction and therefore rises in fetal O₂ consumption. Furthermore, in the "in series" adult circulation, the left ventricular (LV) output is distributed to systemic tissues and is equal to the right ventricular (RV) output, which is distributed to the lungs (32). By contrast, in the "in parallel" fetal circulation 1) RV output is 50-100% greater than the LV output (32, 36) and 2) the left and right ventricles both have a systemic distribution, with the combined ventricular output passing not only to the fetal body but also the placenta, the site of feto-maternal gas exchange (32). Thus an alteration in the balance between the LV and RV outputs, or the relative distribution of blood flow to the fetal body and placenta, may alter O₂ delivery patterns to the fetus and thereby affect O₂ usage by fetal tissues. However, it is unknown if NO influences the relative level of the LV or RV outputs or blood flow distribution between the fetal body and placenta.

Accordingly, the aims of the present study were to evaluate the role of NO in modulating 1) the relative levels of LV and RV outputs, 2) the distribution of the systemic output between the fetal body and placenta, and 3) fetal body O₂ extraction and O₂ consumption. Experiments were performed in chronically instrumented late-gestation fetal lambs, in which hemodynamic, blood flow, and blood gas measurements were performed after incremental inhibition of NO synthesis with the stereospecific and potent NO synthase inhibitor N-nitro-L-arginine (L-NNA).

	MATERIALS AND METHODS

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Experiments were approved by the Monash University Animal Experimentation Committee and conducted in accord with guidelines established by the National Health and Medical Research Council of Australia.

Animal preparation. Eight fetuses with known breeding dates were chronically instrumented under aseptic conditions at 128-134 days gestation (term 147 days). Fasted Border-Leicester cross ewes were anesthetized with propofol (5 mg/kg iv), intubated, and then mechanically ventilated with 1-3% halothane and a 2:1 nitrous oxide-oxygen mixture. The pregnant horn of the uterus was exposed through a midline laparotomy, and the fetal head, left forelimb, and upper thorax were delivered through a hysterotomy. A fetal thoracotomy was performed in the 3rd left interspace, and the 4th rib was removed to increase exposure of the heart and great vessels. After incision of the pericardium, an 8- or 10-mm ultrasonic flow probe (Transonic Systems, Ithaca, NY) was placed around the pulmonary trunk. A Teflon cannula was inserted into the pulmonary trunk through an adventitial purse-string suture distal to the flow probe and connected to a polyvinyl catheter. After insertion of a polyvinyl catheter into the left atrial cavity through a purse-string suture in the appendage, the pericardium was loosely closed and the overlying muscle layers were repaired. The left axillary artery and vein were then exposed, and catheters were passed via these vessels into the brachiocephalic trunk and superior vena cava, respectively. All catheters were exteriorized, and after a wide-bore catheter was sutured to the skin of the anterior chest wall for measurement of amniotic fluid pressure, the fetal skin and maternal uterine incisions were closed.

Experimental protocol. Ewes were placed in a mobile laboratory cart 3-4 days after surgery and allowed free access to feed and water. Fetal brachiocephalic trunk blood pressure, heart rate, and pulmonary trunk flow were recorded, and 0.4-ml blood samples were collected anaerobically from the brachiocephalic trunk, pulmonary trunk, abdominal aorta, and the umbilical vein for hemoglobin and blood gas analysis. Fetal ventricular outputs and blood flows to the fetal body and placenta were then measured with radioactive microspheres using the reference sample method (18). After baseline measurements, NO synthesis was inhibited with the stereospecific NO synthase inhibitor L-NNA (Sigma Chemical), which was dissolved in normal saline to a concentration of 5 mg/ml and infused continuously through the hindlimb venous catheter at a rate of 0.68 mg · kg¹ · min¹ to nominal cumulative doses of 10 and 25 mg/kg, assuming a fetal body weight of 4 kg. At each L-NNA dose, hemodynamics were allowed to stabilize over a 5-min period, and blood pressure, heart rate, blood flow, and blood gas measurements were then repeated.

Physiological measurements. Brachiocephalic trunk blood pressure was referenced to amniotic fluid pressure. Both pressures were monitored with silicon-chip pressure transducers (model CDX 111; COBE Laboratories, Lakewood, CO), which were calibrated against a water manometer before each experiment. Pulmonary trunk flow was measured continuously with an ultrasonic flowmeter (model T208, Transonic Systems). The outputs from the pressure transducers and flowmeter were amplified using a programmable signal conditioner (Cyberamp model 380; Axon Instruments, Foster City, CA), and signals were continuously displayed on a paper recorder (model 800Z; Neomedix Systems, Sydney, Australia). At baseline and the two L-NNA doses, a 30-s segment of hemodynamic data was also digitized with an analog-to-digital converter at a sampling rate of 200 Hz and stored on computer hard disk for subsequent off-line analysis using customized interactive software.

Radioactive microsphere technique. Radioactive microspheres, 15 µm in diameter and labeled with one of five gamma-emitting isotopes (¹⁴¹Ce, ¹¹³Sn, ⁸⁵Sr, ⁹⁵Nb, or ⁴⁶Sc; NEN, Boston, MA) were ultrasonicated for 10-15 min before injection and injected over 30 to 45 s with 5 ml isotonic saline. At baseline and at an L-NNA dose of 25 mg/kg, two different microsphere labels were injected simultaneously, one into the left atrium to measure LV output and the other into the superior vena cava to measure RV output (37), whereas reference samples were drawn simultaneously from the pulmonary trunk, brachiocephalic trunk, and descending aorta for determination of fetal body and umbilical-placental flows. At an L-NNA dose of 10 mg/kg, LV output was obtained with a single microsphere label injected into the left atrium, whereas reference samples were withdrawn from the brachiocephalic trunk and descending aorta to obtain fetal body and placental flows. Approximately 0.5-1 × 10⁶ microspheres were injected per radiolabel, and reference samples were withdrawn at a rate of 4.1 ml/min with a mechanical pump (model 901A; Harvard Apparatus, South Natick, MA). Reference sample collection was commenced 5-10 s before injection of microspheres and continued for an additional 75 s after the end of injection. Blood withdrawn in the reference samples was simultaneously replaced with maternal blood .

Calculation of fetal ventricular outputs, fetal body, and placental flows. Radioactive microsphere blood flow measurements were calculated using the general relation _Tissue = (_Reference × R_Tissue)/R_Reference, where is flow (ml/min) and R is radioactivity (counts/min). LV and RV outputs and systemic flows were obtained using an adaptation of this general relation for the fetal circulation (37). Thus fetal LV output (_LV) was equal to (_Reference × R_LA)/R_LABCT, where R_LA is the radioactivity of the label injected into the left atrial cavity and R_LABCT is the radioactivity of the same label collected in the brachiocephalic trunk reference sample. Fetal RV output (_RV) was equivalent to (_Reference × R_RV)/R_VPT, where R_RV is the radioactivity of venous label passing into the right ventricle, calculated as the injected radioactivity of this label minus that portion crossing the foramen ovale to appear in the LV output and R_VPT is the radioactivity of the venous label in the pulmonary reference sample (37). RV output at an L-NNA dose of 10 mg/kg was obtained by interpolation from the measured pulmonary trunk flow, using baseline and 25 mg/kg L-NNA ultrasonic flow probe measurements of pulmonary trunk flow and radioactive microsphere determinations of RV output.

Blood gas calculations. The O₂ content of arterial or venous blood (ml O₂ per dl blood) was calculated as (1.36 × HbS × Hb/100) + (0.003 × PO₂), where HbS is hemoglobin O₂ saturation (%), Hb is hemoglobin level (g/dl), and PO₂ is O₂ tension (mmHg).

Statistics. Changes in hemodynamics, blood flows, and blood gas variables were analyzed with repeated-measures one-way ANOVA (38). The sums of squares were partitioned into individual degrees of freedom, and the significance of changes was evaluated using the Bonferroni procedure, as appropriate, for multiple tests (41). Results are reported as means ± SE, and P < 0.05 was considered significant.

	RESULTS

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Fetoplacental weight was 3.9 ± 0.2 kg, so that the nominal doses of 10 and 25 mg/kg L-NNA corresponded to infusion rates of 10.5 ± 0.5 and 26.3 ± 1.3 mg/kg, respectively.

Hemodynamics and blood gases. Mean brachiocephalic trunk blood pressure increased by 13.5 ± 1.5 mmHg (P < 0.005), and heart rate fell by 20 ± 3 beats/min (P < 0.005) after administration of 10 mg/kg L-NNA, but neither changed further at 25 mg/kg L-NNA dose. With the exception of an increase in hemoglobin concentration (P < 0.005) and a reduction in pH (P < 0.05), blood gas variables in the brachiocephalic trunk were unchanged between baseline and 10 mg/kg L-NNA. The higher dose of L-NNA was accompanied by a further rise in hemoglobin concentration (P < 0.025) and a fall in pH (P < 0.025), whereas PCO₂ increased (P < 0.005) and hemoglobin O₂ saturation decreased (P < 0.005) compared with the baseline level. Neither PO₂ nor O₂ content changed significantly with L-NNA (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Ascending aortic hemodynamics and blood gases before and after N -nitro-L-arginine

Ventricular outputs. The baseline combined ventricular output was 505 ± 16 ml · min¹ · kg¹ with the contribution of LV output (41.3 ± 1.4%) being less than RV output (58.7 ± 1.4%, P < 0.005). The combined ventricular output fell to 405 ± 11 ml · min¹ · kg¹ at 10 mg/kg L-NNA (P < 0.005), and then declined further to 350 ± 19 ml · min¹ · kg¹ at 25 mg/kg L-NNA (P < 0.05, Fig. 1A). However, the proportional contribution of the left and right ventricles to the combined ventricular output was unchanged between baseline and 10 and 25 mg/kg L-NNA (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Changes in combined ventricular output (A) and proportion of combined ventricular output constituted by left (open bars) and right (filled bars) ventricular outputs (B) at baseline and after 10 and 25 mg/kg N-nitro-L-arginine (L-NNA). *** P < 0.005 left vs. right ventricular output.

Fetal body and placental blood flows. Baseline fetal body blood flow (330 ± 19 ml · min¹ · kg¹) fell to 228 ± 16 ml · min¹ · kg¹ at 10 mg/kg L-NNA (P < 0.005) and was not statistically different at 25 mg/kg L-NNA (Fig. 2A). By contrast, placental blood flow was unchanged between baseline (175 ± 9 ml · min¹ · kg¹) and 10 mg/kg L-NNA (177 ± 12 ml · min¹ · kg¹), but fell to 140 ± 9 ml · min¹ · kg¹ at 25 mg/kg L-NNA (P < 0.005, Fig. 2B). As a result of these divergent changes, the ratio of fetal body to placental blood flow fell from 1.93 ± 0.17 at baseline to 1.40 ± 0.23 at 10 mg/kg L-NNA (P < 0.05) and was not significantly different at 25 mg/kg L-NNA (1.53 ± 0.11; Fig. 2C).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Changes in fetal body (A) and placental (B) blood flow and fetal body-to-placental blood flow ratio (C) at baseline and after 10 and 25 mg/kg L-NNA.

Fetal body arterial and venous O₂ contents. Average arterial fetal body O₂ content was 6.3 ± 0.2 ml/dl at baseline and was unaltered at both 10 and 25 mg/kg L-NNA (Fig. 3A). By contrast, the calculated average venous O₂ content fell from 4.3 ± 0.2 ml/dl at baseline to 2.7 ± 0.5 ml/dl at 10 mg/kg L-NNA (P < 0.005) and was not statistically different at 25 mg/kg (Fig. 3B). As a result, fetal body O₂ extraction increased from 2.1 ± 0.2 ml/dl to 3.6 ± 0.4 ml/dl at 10 mg/kg L-NNA (P < 0.005), and did not change further at 25 mg/kg L-NNA (Fig. 3C).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Changes in average fetal arterial (A) and venous (B) O2 contents and fetal O2 extraction (C) at baseline and after 10 and 25 mg/kg L-NNA.

Fetal body O₂ delivery, O₂ consumption, and O₂ extraction coefficient Average fetal body O₂ delivery fell from 20.6 ± 0.9 ml · min¹ · kg¹ at baseline to 14.4 ± 1.3 ml · min¹ · kg¹ at 10 mg/kg L-NNA (P < 0.005) and did not fall further at 25 mg/kg L-NNA (Fig. 4A). By contrast, fetal body whole body O₂ consumption increased from 6.7 ± 0.4 to 7.7 ± 0.5 ml · min¹ · kg¹ between baseline and 10 mg/kg L-NNA (P < 0.05) and then fell to a near baseline value of 6.4 ± 0.5 ml · min¹ · kg¹ at 25 mg/kg L-NNA (P < 0.01, Fig. 4B). These changes in whole body O₂ consumption were accompanied by alterations in the fetal body O₂ extraction coefficient, which increased from 0.33 ± 0.03 at baseline to 0.58 ± 0.08 at 10 mg/kg L-NNA (P < 0.005) and was not altered further at 25 mg/kg L-NNA (Fig. 4C).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Changes in average fetal O2 delivery (A), consumption (B), and extraction coefficient (C) at baseline and after 10 and 25 mg/kg L-NNA.

	DISCUSSION

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Three main findings have emerged from this study, which has examined the effect of NO synthase inhibition on ventricular outputs, fetoplacental blood flow distribution, as well as whole body O₂ extraction and O₂ consumption in chronically instrumented late-gestation fetal sheep. First, whereas NO synthase inhibition reduced the fetal combined ventricular output, it did not alter the relative balance between the LV and RV outputs. Second, because a reduction in fetal body blood flow preceded and was of greater magnitude than a decrease in placental blood flow, NO synthase inhibition was associated with a systemic flow redistribution away from the fetal body and toward the placenta. Last, despite a fall in fetal body perfusion, NO synthase inhibition was accompanied by a proportionally greater increase in fetal body O₂ extraction, which initially supported a rise in fetal whole body O₂ consumption and subsequently maintained fetal whole body O₂ consumption at near baseline levels after a reduction in placental perfusion.

Previous studies addressing the role of NO in the fetal circulation have generally examined hemodynamic and blood gas responses at single dose of NO synthase inhibitor (5, 27). The use of an incremental infusion regimen in this study has pointed to a number of dose-related differences between the responses of hemodynamic blood flow and blood gas variables to fetal NO synthase inhibition. Thus a rise in blood pressure associated with NO synthase inhibition attained a plateau by 10 mg/kg L-NNA. Given the wide acceptance of the concept that blood vessel tone represents the overall balance between circulating and locally released vasodilator and vasoconstrictor influences acting on the vessel wall (26), this increase in blood pressure was most likely related to the loss of a significant endogenous vasodilator mechanism counteracting the vasoconstrictor effects of the sympathetic nervous system (24) and circulating agents such as endothelin-1 (1, 6), ANG II (21), and norepinephrine (7). Inhibition of NO synthase was also accompanied by a fall in heart rate which, because baroreceptor responses are present in the late-gestation fetus (24), most likely represented a reflex response to the rise in blood pressure.

Inhibition of NO synthase in the present study also resulted in a progressive rise in hemoglobin concentration. Because no significant release of erythrocytes occurs from storage sites such as the spleen in chronically instrumented fetal sheep (2), the most plausible explanation for this finding was a reduction in plasma volume associated with a fluid shift from intravascular to extravascular compartments. One factor that presumably contributed to such a fluid shift was the high permeability of fetal capillaries, which predisposes to transudation of fluid across the capillary membrane with increases in hydrostatic pressure (3). Consistent with this proposal, increases in hemoglobin concentration accompanied by falls in fetal blood volume (6, 7, 21) have been reported after elevation of fetal blood pressure via infusion of vasoconstrictor compounds such as endothelin-1 (1, 6), norepinephrine (7, 29), epinephrine (29), or ANG II (29). However, the presence of a progressive rise in hemoglobin concentration in the face of similar blood pressure levels at 10 and 25 mg/kg L-NNA (Table 1) suggests that NO synthase inhibition may have also directly augmented vascular permeability (22).

The observed changes in fetal blood pressure, heart rate, and hemoglobin concentration that accompanied inhibition of NO synthase are particularly relevant because increases in arterial blood pressure (17, 40), reductions in heart rate (32), hemoconcentration (12), and falls in circulating blood volume (14) all reduce fetal cardiac output in the fetus. It is therefore likely that these alterations contributed to the fall in combined ventricular output evident with inhibition of NO synthesis in the present study. On the other hand, the contribution of the left and right ventricles to the combined ventricular output were unaffected after L-NNA. Taken together, these results suggest that, via its hemodynamic effects, NO supports the maintenance of a high level of cardiac output characteristic of the fetal circulation (32) but is not involved in the regulation of the relative magnitudes of the fetal LV and RV outputs.

In this study, a decline in combined ventricular output evident at 10 mg/kg L-NNA was related to a fall in fetal body blood flow, whereas a further decline in combined ventricular output apparent at 25 mg/kg L-NNA was predominantly due to a reduction in placental perfusion. It is likely that this differing pattern of blood flow changes, which effectively resulted in a redistribution of blood flow away from the fetal body and toward the placental compartment, was related to at least three factors. The first was that the sensitivity of NO synthase to inhibition with L-NNA was greater in the fetal body than in the placenta, a scenario that is compatible with the recent report that gene expression of the inducible form of NO synthase is normally present in the fetal circulation and is particularly prominent in the placenta (4), and the pharmacological evidence pointing to a greater effect of L-NNA on constitutive compared with inducible NO synthase (13, 28). The second factor is that basal release of NO accounted for a lesser portion of the vasodilator influence in the placenta compared with the fetal body, a proposal in accord with observations suggesting that the vasodilatory role of NO diminishes in late gestation in the umbilical-placental circulation (35). Finally, it is possible that vasoconstriction after inhibition of NO synthesis was less pronounced in the placenta than in the fetal body because of the absence of innervation of the umbilical and placental vessels (30) and the consequent lack of a tonic sympathetic neural influence.

Despite the absence of any significant changes in arterial hemoglobin O₂ saturation, PO₂ and O₂ content, the initial dose of 10 mg/kg L-NNA had striking effects on fetal whole body oxygenation variables in the present study. Thus O₂ delivery to fetal body tissues fell by 30%, a change that was entirely attributable to a reduction in fetal body blood flow. Moreover, the mixed venous O₂ content fell markedly in association with a marked increase in systemic O₂ extraction and a rise in the O₂ extraction coefficient to a level (0.58) that was even greater than the 0.53 attained with a 50% umbilical blood flow reduction produced via cord occlusion (19). Importantly, however, the magnitude of the increase in fetal systemic O₂ extraction after 10 mg/kg L-NNA exceeded the fall in fetal body blood flow, so that fetal whole body O₂ consumption increased by 15%. The latter finding is consistent with the notion that, as in the adult (34), constitutive production of NO has an inhibitory effect on fetal oxidative metabolism. However, two important differences were evident between fetal and adult whole body O₂ consumption data following NO synthase inhibition. First, the magnitude of the increase in whole body O₂ consumption in the present study was about one-half the increase (27%) seen in adult dogs (34), a difference primarily related to a rise in O₂ extraction (134%) that was nearly double the 71% increment observed in fetal lambs. Second, the stimulatory effect of NO inhibition on whole body O₂ consumption in the fetus appeared to depend on the preservation of placental perfusion, because in the presence of a reduced placental blood flow evident at 25 mg/kg L-NNA, a still-present increase in O₂ extraction then served to maintain whole body O₂ consumption at a near baseline level.

The pattern of changes in fetal whole body O₂ extraction and O₂ consumption occurring in this study in the setting of a reduction in both the combined ventricular output and fetal body blood flow cannot be readily accounted for by hemodynamic or neurohumoral factors. Thus the increases in fetal whole body O₂ extraction and consumption were unlikely to have been related to systemic vasoconstriction per se, because infusion of vasoconstrictors such as endothelin-1 is associated with an unchanged fetal O₂ extraction and a decrease in whole body O₂ consumption (1). Furthermore, although infusion of the sympathetic neurotransmitter norepinephrine can elevate fetal whole body O₂ extraction and consumption (25), the increases in the latter variables observed in the present study were unlikely to have been related to sympathetic mechanisms because evidence from adult rabbits indicates that NO synthase inhibition is associated with a baroceptor-mediated decrease in sympathetic nerve activity (15). Accordingly, the pattern of changes in fetal whole body O₂ extraction and O₂ consumption observed in this study appear to be specific to NO synthase inhibition.

The circulatory responses that occurred with NO synthase inhibition are of particular interest because they resemble the hemodynamic and metabolic changes observed during fetal hypoxemia in a number of respects. Thus acute hypoxemia results in bradycardia and hypertension (9, 31), an increase in systemic O₂ extraction which serves to maintain fetal O₂ consumption (10) and, if severe enough, a redistribution of cardiac output from fetal body to placenta (9). The hypertension appears in large part to be related to hypoxia-induced increases in the circulating levels of a range of vasoconstrictor compounds, including norepinephrine (8), endothelin-1 (16), vasopressin (33), as well as ACTH and cortisol (20). The increase in circulating levels of norepinephrine occurring in fetal hypoxemia may also augment systemic O₂ extraction (25). However, as NO also requires molecular O₂ for its formation (23), it is possible that in the low O₂ environment of the fetus the level of tissue oxygenation may be a critical limiting step in NO production. With a diminution in tissue O₂ delivery, a reduction in NO production may therefore constitute an additional mechanism contributing to a redistribution of blood flow from the fetal body to the placenta and, via disinhibition of fetal oxidative metabolism, an increase in O₂ extraction.

In conclusion, in the fetus, inhibition of NO synthesis results in a reduction in the combined ventricular output, a redistribution of systemic blood flow to favor the placenta, and an increase in fetal body O₂ extraction, which initially increases whole body O₂ consumption and then maintains whole body O₂ consumption at near baseline levels following a fall in placental perfusion.