Inhibition of 20-HETE abolishes the myogenic response during NOS antagonism in the ovine fetal pulmonary circulation

Mechanisms that maintain high pulmonary vascular resistance (PVR) and oppose vasodilation in the fetal lung are poorly understood. In fetal lambs, increased pulmonary artery pressure evokes a potent vasoconstriction, suggesting that a myogenic response contributes to high PVR in the fetus. In adult systemic circulations, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to modulate the myogenic response, but its role in the fetal lung is unknown. We hypothesized that acute increases in pulmonary artery pressure release 20-HETE, which causes vasoconstriction, or a myogenic response, in the fetal lung. To address this hypothesis, we studied the hemodynamic effects of N-methylsufonyl-12,12-dibromododec-11-enamide (DDMS), a specific inhibitor of 20-HETE production, on the pulmonary vasoconstriction caused by acute compression of the ductus arteriosus (DA) in chronically prepared fetal sheep. An inflatable vascular occluder around the DA was used to increase pulmonary artery pressure under three study conditions: control, after pretreatment with nitro-L-arginine (L-NA; to inhibit shear-stress vasodilation), and after combined treatment with both L-NA and a specific 20-HETE inhibitor, DDMS. We found that DA compression after L-NA treatment increased PVR by 44 +/- 12%. Although intrapulmonary DDMS infusion did not affect basal PVR, DDMS completely abolished the vasoconstrictor response to DA compression in the presence of L-NA (44 +/- 12% vs. 2 +/- 4% change in PVR, L-NA vs. L-NA + DDMS, P < 0.05). We conclude that 20-HETE mediates the myogenic response in the fetal pulmonary circulation and speculate that pharmacological inhibition of 20-HETE might have a therapeutic role in neonatal conditions characterized by pulmonary hypertension.     

Inhaled carbon monoxide does not cause pulmonary vasodilation in the late-gestation fetal lamb

As observed with nitric oxide (NO), carbon monoxide (CO) binds and may activate soluble guanylate cyclase and increase cGMP levels in smooth muscle cells in vitro. Because inhaled NO (I(NO)) causes potent and sustained pulmonary vasodilation, we hypothesized that inhaled CO (I(CO)) may have similar effects on the perinatal lung. To determine whether I(CO) can lower pulmonary vascular resistance (PVR) during the perinatal period, we studied the effects of I(CO) on late-gestation fetal lambs. Catheters were placed in the main pulmonary artery, left pulmonary artery (LPA), aorta, and left atrium to measure pressure. An ultrasonic flow transducer was placed on the LPA to measure blood flow to the left lung. After baseline measurements, fetal lambs were mechanically ventilated with a hypoxic gas mixture (inspired O(2) fraction < 0.10) to maintain a constant fetal arterial PO(2). After 60 min (baseline), the lambs were treated with I(CO) [5-2,500 parts/million (ppm)]. Comparisons were made with I(NO) (5 and 20 ppm) and combined I(NO) (5 ppm) and I(CO) (100 and 2,500 ppm). We found that I(CO) did not alter left lung blood flow or PVR at any of the study doses. In contrast, low-dose I(NO) decreased PVR by 47% (P < 0.005). The combination of I(NO) and I(CO) did not enhance the vasodilator response to I(NO). To determine whether endogenous CO contributes to vascular tone in the fetal lung, zinc protoporphyrin IX, an inhibitor of heme oxygenase, was infused into the LPA in three lambs. Zinc protoporphyrin IX had no effect on baseline PVR, aortic pressure, or the pressure gradient across the ductus arteriosus. We conclude that I(CO) does not cause vasodilation in the near-term ovine transitional circulation, and endogenous CO does not contribute significantly to baseline pulmonary vascular tone or ductus arteriosus tone in the late-gestation ovine fetus.     

Role of neuronal nitric oxide synthase in regulation of vascular and ductus arteriosus tone in the ovine fetus

Nitric oxide (NO) is produced by NO synthase (NOS) and contributes to the regulation of vascular tone in the perinatal lung. Although the neuronal or type I NOS (NOS I) isoform has been identified in the fetal lung, it is not known whether NO produced by the NOS I isoform plays a role in fetal pulmonary vasoregulation. To study the potential contribution of NOS I in the regulation of basal fetal pulmonary vascular resistance (PVR), we studied the hemodynamic effects of a selective NOS I antagonist, 7-nitroindazole (7-NINA), and a nonselective NOS antagonist, N-nitro-L-arginine (L-NNA), in chronically prepared fetal lambs (mean age 128 +/- 3 days, term 147 days). Brief intrapulmonary infusions of 7-NINA (1 mg) increased basal PVR by 37% (P < 0.05). The maximum increase in PVR occurred within 20 min after infusion, and PVR remained elevated for up to 60 min. Treatment with 7-NINA also increased the pressure gradient between the pulmonary artery and aorta, suggesting constriction of the ductus arteriosus (DA). To test whether 7-NINA treatment selectively inhibits the NOS I isoform, we studied the effects of 7-NINA and L-NNA on acetylcholine-induced pulmonary vasodilation. The vasodilator response to acetylcholine remained intact after treatment with 7-NINA but was completely inhibited after L-NNA, suggesting minimal effects on endothelial or type III NOS after 7-NINA infusion. Western blot analysis detected NOS I protein in the fetal lung and great vessels including the DA. NOS I protein was detected in intact and endothelium-denuded vessels, suggesting that NOS I is present in the medial or adventitial layer. We conclude that 7-NINA, a selective NOS I antagonist, increases basal PVR, systemic arterial pressure, and DA tone in the late-gestation fetus and that NOS I protein is present in the fetal lung and great vessels. We speculate that NOS I may contribute to NO production in the regulation of basal vascular tone in the pulmonary and systemic circulations and the DA.     

Contribution of endothelin to pulmonary vascular tone under normoxic and hypoxic conditions

The contribution of endothelin to resting pulmonary vascular tone and hypoxic pulmonary vasoconstriction in humans is unknown. We studied the hemodynamic effects of BQ-123, an endothelin type A receptor antagonist, on healthy volunteers exposed to normoxia and hypoxia. Hemodynamics were measured at room air and after 15 min of exposure to hypoxia (arterial PO(2) 99.8 +/- 1.8 and 49.4 +/- 0.4 mmHg, respectively). Measurements were then repeated in the presence of BQ-123. BQ-123 decreased pulmonary vascular resistance (PVR) 26% and systemic vascular resistance (SVR) 21%, whereas it increased cardiac output (CO) 22% (all P < 0.05). Hypoxia raised CO 28% and PVR 95%, whereas it reduced SVR 23% (all P < 0.01). During BQ-123 infusion, hypoxia increased CO 29% and PVR 97% and decreased SVR 22% (all P < 0.01). The pulmonary vasoconstrictive response to hypoxia was similar in the absence and presence of BQ-123 [P = not significant (NS)]. In vehicle-treated control subjects, hypoxic pulmonary vasoconstriction did not change with repeated exposure to hypoxia (P = NS). Endothelin contributes to basal pulmonary and systemic vascular tone during normoxia, but does not mediate the additional pulmonary vasoconstriction induced by acute hypoxia.     

Effects of chronic estrogen-receptor blockade on ovine perinatal pulmonary circulation

Prolonged infusions of 17beta-estradiol reduce fetal pulmonary vascular resistance (PVR), but the effects of endogenous estrogens in the fetal pulmonary circulation are unknown. To test the hypothesis that endogenous estrogen promotes pulmonary vasodilation at birth, we studied the hemodynamic effects of prolonged estrogen-receptor blockade during late gestation and at birth in fetal lambs. We treated chronically prepared fetal lambs with ICI-182,780 (ICI, a specific estrogen-receptor blocker, n = 5) or 1% DMSO (CTRL, n = 5) for 7 days and then measured pulmonary hemodynamic responses to ventilation with low- and high-fraction inspired oxygen (FI(O(2))). Treatment with ICI did not change basal fetal PVR or arterial blood gas tensions. However, treatment with ICI abolished the vasodilator response to ventilation with low FI(O(2)) [change in PVR -30 +/- 6% (CTRL) vs. +10 +/- 13%, (ICI), P < 0.05] without reducing the vasodilator response to ventilation with high FI(O(2)) [change in PVR, -73 +/- 3% (CTRL) vs. -77 +/- 4%, (ICI); P = not significant]. ICI treatment reduced prostacyclin synthase (PGIS) expression by 33% (P < 0.05) without altering expression of endothelial nitric oxide synthase or cyclooxygenase-1 and -2. In situ hybridization and immunohistochemistry revealed that PGIS is predominantly expressed in the airway epithelium of late gestation fetal lambs. We conclude that prolonged estrogen-receptor blockade inhibits the pulmonary vasodilator response at birth and that this effect may be mediated by downregulation of PGIS. We speculate that estrogen exposure during late gestation prepares the pulmonary circulation for postnatal adaptation.     

Pulmonary vascular effects of sildenafil on the development of chronic pulmonary hypertension in the ovine fetus

We investigated the pulmonary vascular effects of prophylactic use of sildenafil, a specific phosphodiesterase-5 inhibitor, in late-gestation fetal lambs with chronic pulmonary hypertension. Fetal lambs were operated on at 129 +/- 1 days gestation (term = 147 days). Ductus arteriosus (DA) was compressed for 8 days to cause chronic pulmonary hypertension. Fetuses were treated with sildenafil (24 mg/day) or saline. Pulmonary vascular responses to increase in shear stress and in fetal PaO2 were studied at, respectively, day 4 and 6. Percent wall thickness of small pulmonary arteries (%WT) and the right ventricle-to-left ventricle plus septum ratio (RVH) were measured after completion of the study. In the control group, DA compression increased PA pressure (48 +/- 5 to 72 +/- 8 mmHg, P < 0.01) and pulmonary vascular resistance (PVR) (0.62 +/- 0.08 to 1.15 +/- 0.11 mmHg x ml(-1) x min(-1), P < 0.05). Similar increase in PAP was observed in the sildenafil group, but PVR did not change significantly (0.54 +/- 0.06 to 0.64 +/- 0.09 mmHg x ml(-1) x min(-1)). Acute DA compression, after brief decompression, elevated PVR 25% in controls and decreased PVR 35% in the sildenafil group. Increased fetal PaO2 did not change PVR in controls but decreased PVR 60% in the sildenafil group. %WT and RVH were not different between groups. Prophylactic sildenafil treatment prevents the rise in pulmonary vascular tone and altered vasoreactivity caused by DA compression in fetal lambs. These results support the hypothesis that elevated PDE5 activity is involved in the consequences of chronic pulmonary hypertension in the perinatal lung.     

Fasudil inhibits the myogenic response in the fetal pulmonary circulation

In addition to high pulmonary vascular resistance (PVR) and low pulmonary blood flow, the fetal pulmonary circulation is characterized by mechanisms that oppose vasodilation. Past work suggests that high myogenic tone contributes to high PVR and may contribute to autoregulation of blood flow in the fetal lung. Rho-kinase (ROCK) can mediate the myogenic response in the adult systemic circulation, but whether high ROCK activity contributes to the myogenic response and modulates time-dependent vasodilation in the developing lung circulation are unknown. We studied the effects of fasudil, a ROCK inhibitor, on the hemodynamic response during acute compression of the ductus arteriosus (DA) in chronically prepared, late-gestation fetal sheep. Acute DA compression simultaneously induces two opposing responses: 1) blood flow-induced vasodilation through increased shear stress that is mediated by NO release and 2) stretch-induced vasoconstriction (i.e., the myogenic response). The myogenic response was assessed during acute DA compression after treatment with N(omega)-nitro-L-arginine, an inhibitor of nitric oxide synthase, to block flow-induced vasodilation and unmask the myogenic response. Intrapulmonary fasudil infusion (100 microg over 10 min) did not enhance flow-induced vasodilation during brief DA compression but reduced the myogenic response by 90% (P<0.05). During prolonged DA compression, fasudil prevented the time-dependent decline in left pulmonary artery blood flow at 2 h (183+/-29 vs. 110+/-11 ml/min with and without fasudil, respectively; P<0.001). We conclude that high ROCK activity opposes pulmonary vasodilation in utero and that the myogenic response maintains high PVR in the normal fetal lung through ROCK activation.     

Pulmonary vascular dysfunction in preterm lambs with chronic lung disease

Chronic lung injury from prolonged mechanical ventilation after premature birth inhibits the normal postnatal decrease in pulmonary vascular resistance (PVR) and leads to structural abnormalities of the lung circulation in newborn sheep. Compared with normal lambs born at term, chronically ventilated preterm lambs have increased pulmonary arterial smooth muscle and elastin, fewer lung microvessels, and reduced abundance of endothelial nitric oxide synthase. These abnormalities may contribute to impaired respiratory gas exchange that often exists in infants with chronic lung disease (CLD). Nitric oxide inhalation (iNO) reduces PVR in human infants and lambs with persistent pulmonary hypertension. We wondered whether iNO might have a similar effect in lambs with CLD. We therefore studied the effect of iNO on PVR in lambs that were delivered prematurely at approximately 125 days of gestation (term = 147 days) and mechanically ventilated for 3 wk. All of the lambs had chronically implanted catheters for measurement of pulmonary vascular pressures and blood flow. During week 2 of mechanical ventilation, iNO at 15 parts/million for 1 h decreased PVR by approximately 20% in 12 lambs with evolving CLD. When the same study was repeated in eight lambs at the end of week 3, iNO had no significant effect on PVR. To see whether this loss of iNO effect on PVR might reflect dysfunction of lung vascular smooth muscle, we infused 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP; 150 microg. kg(-1). min(-1) iv) for 15-30 min in four of these lambs at the end of week 3. PVR consistently decreased by 30-35%. Lung immunohistochemistry and immunoblot analysis of excised pulmonary arteries from lambs with CLD, compared with control term lambs, showed decreased soluble guanylate cyclase (sGC). These results suggest that loss of pulmonary vascular responsiveness to iNO in preterm lambs with CLD results from impaired signaling, possibly related to deficient or defective activation of sGC, the intermediary enzyme through which iNO induces increased vascular smooth muscle cell cGMP and resultant vasodilation.     

Endothelial alterations during inhaled NO in lambs with pulmonary hypertension: implications for rebound hypertension

Clinically significant increases in pulmonary vascular resistance (PVR) have been noted upon acute withdrawal of inhaled nitric oxide (iNO). Previous studies in the normal pulmonary circulation demonstrate that iNO increases endothelin-1 (ET-1) levels and decreases endogenous nitric oxide synthase (NOS) activity, implicating an endothelial etiology for the increase in resistance upon iNO withdrawal. However, the effect of iNO on endogenous endothelial function in the clinically relevant pulmonary hypertensive circulation is unknown. The objective of this study was to determine the effects of iNO on endogenous NO-cGMP and ET-1 signaling in lambs with preexisting pulmonary hypertension secondary to increased pulmonary blood flow. Eight fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt lambs). After delivery (4 wk), the shunt lambs were mechanically ventilated with iNO (40 ppm) for 24 h. After 24 h of inhaled NO, plasma ET-1 levels increased by 34.8% independently of changes in protein levels (P < 0.05). Contrary to findings in normal lambs, total NOS activity did not decrease during iNO. In fact, Western blot analysis demonstrated that tissue endothelial NOS protein levels decreased by 43% such that NOS activity relative to protein levels actually increased during iNO (P < 0.05). In addition, the beta-subunit of soluble guanylate cyclase decreased by 70%, whereas phosphodiesterase 5 levels were unchanged (P < 0.05). Withdrawal of iNO was associated with an acute increase in PVR, which exceeded baseline PVR by 45%, and a decrease in cGMP concentrations to levels that were below baseline. These data suggest that the endothelial response to iNO and the potential mechanisms of rebound pulmonary hypertension are dependent upon the underlying pulmonary vasculature.     

EDRF inhibition attenuates the increase in pulmonary blood flow due to oxygen ventilation in fetal lambs.

At birth, pulmonary vasodilation occurs during rhythmic distension of the lungs and oxygenation. Inhibition of prostaglandin synthesis prevents pulmonary vasodilation during rhythmic distension of the lungs but not during oxygenation. Because endothelium-derived relaxing factor (EDRF) modulates pulmonary vascular tone at birth, at rest, and during hypoxia in older animals, we hypothesized that EDRF may modulate pulmonary vascular tone during oxygenation in fetal lambs. We studied the responses to N omega-nitro-L-arginine, a competitive inhibitor of EDRF synthesis, in nine near-term fetal lambs and to drug vehicle in six of these lambs and the subsequent responses to in utero ventilation with 95% O2 in these fetal lambs. In all fetal lambs, prostaglandin synthesis was prevented by meclofenamate. N omega-nitro-L-arginine increased pulmonary and systemic arterial pressures by 28% (P < 0.05) and 31% (P < 0.05), respectively, and decreased pulmonary blood flow by 83% (P < 0.05). In the controls, ventilation with 95% O2 increased pulmonary blood flow by 1,050% (P = 0.05) without changing pressures, thereby decreasing pulmonary vascular resistance by 88% (P = 0.05). During N omega-nitro-L-arginine infusion, ventilation with 95% O2 increased pulmonary blood flow by 162% (P = 0.05) and decreased pulmonary vascular resistance by 74% (P = 0.05). This suggests that EDRF may play an important role in modulating resting pulmonary vascular tone in fetal lambs and in the vasodilatory response to ventilation with O2 in utero.     

In vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue.

In vitro evidence suggests that resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation are mediated by changes in vascular smooth muscle concentrations of guanosine 3',5'-cyclic monophosphate (cGMP). We investigated this hypothesis in vivo in 19 mechanically ventilated intact lambs by determining the hemodynamic effects of methylene blue (a guanylate cyclase inhibitor) and then by comparing the hemodynamic response to five vasodilators during pulmonary hypertension induced by the infusion of U-46619 (a thromboxane A2 mimic) or methylene blue. Methylene blue caused a significant time-dependent increase in pulmonary arterial pressure. During U-46619 infusions, acetylcholine, ATP-MgCl2, sodium nitroprusside, isoproterenol, and 8-bromo-cGMP decreased pulmonary arterial pressure. During methylene blue infusions, the decreases in pulmonary arterial pressure caused by acetylcholine and ATP-MgCl2 (endothelium-dependent vasodilators) and sodium nitroprusside (an endothelium-independent guanylate cyclase-dependent vasodilator) were attenuated by greater than 50%. The decreases in pulmonary arterial pressure caused by isoproterenol and 8-bromo-cGMP (endothelium-independent vasodilators) were unchanged. This study in intact lambs supports the in vitro evidence that changes in vascular smooth muscle cell concentrations of cGMP in part mediate resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation.     

Hemodynamic effects of postpulmonary administration of prostaglandin D2 in fetal animals

Dose-response relationships in pulmonary vascular resistance (PVR), mean systemic arterial pressure (SAP), and heart rate (HR) to left atrial administration of prostaglandin D2 (PGD2) were determined in five fetal lambs. Fetuses were delivered by cesarean section from chloralose anesthetized ewes with the umbilical circulation maintained intact. Fetuses were prevented from breathing thus maintaining pulmonary vascular tone in the elevated fetal state. Blood was withdrawn from the inferior vena cava and pumped at constant flow into the lower left lobe of the fetal lung. Postpulmonary infusions of PGD2 brought about dose-dependent decreases in pulmonary vascular resistance. Heart rate tended to increase in fetal lambs. Mean systemic arterial pressure increased in the fetal lambs at all doses tested except for the largest dose (44.14 micrograms/kg X min), which produced slight hypotension. These data demonstrate that exposure to the systemic circulation prior to entering the pulmonary vasculature does not alter the preferential dilator action of PGD2 on fetal pulmonary vessels nor does it produce significant systemic hypotension.     

Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung

Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15-500 microg) and HA-1077 (500 microg), in chronically prepared late-gestation fetal lambs (n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 +/- 0.18 to 0.16 +/- 0.02 mmHg x ml(-1) x min(-1) (P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 +/- 0.03 to 0.19 +/- 0.03 (P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-L-arginine (L-NA; 15-30 mg), a selective NO synthase antagonist. L-NA increased PVR by 127 +/- 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 (P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension.     

NO and prostaglandin interactions during hemodynamic stress in the fetal ovine pulmonary circulation

Nitric oxide (NO) and prostacyclin (PGI(2)) are potent fetal pulmonary vasodilators, but their relative roles and interactions in the regulation of the perinatal pulmonary circulation are poorly understood. We compared the separate and combined effects of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibition during acute hemodynamic stress caused by brief mechanical compression of the ductus arteriosus (DA) in chronically prepared fetal lambs. Nitro-L-arginine (L-NNA; NOS antagonist), meclofenamate (Mec; COX inhibitor), combined drugs (L-NNA-Mec), or saline (control) was infused into the left pulmonary artery (LPA) before DA compression. In controls, DA compression decreased pulmonary vascular resistance (PVR) by 43% (P < 0.01). L-NNA, but not Mec, treatment completely blocked vasodilation and caused a paradoxical increase in PVR (+31%; P < 0.05). The effects of L-NNA-Mec and L-NNA on PVR were similar. To determine if the vasodilator effect of PGI(2) is partly mediated by NO release, we studied PGI(2)-induced vasodilation before and after NOS inhibition. L-NNA treatment blocked the PGI(2)-induced rise in LPA blood flow by 73% (P < 0.001). We conclude that NO has a greater role than PGs in fetal pulmonary vasoregulation during acute hemodynamic stress and that PGI(2)-induced pulmonary vasodilation is largely mediated by NO release in the fetal lung.     

Estradiol improves pulmonary hemodynamics and vascular remodeling in perinatal pulmonary hypertension

Partial ligation of the ductus arteriosus (DA) in the fetal lamb causes sustained elevation of pulmonary vascular resistance (PVR) and hypertensive structural changes in small pulmonary arteries, providing an animal model for persistent pulmonary hypertension of the newborn. Based on its vasodilator and antimitogenic properties in other experimental studies, we hypothesized that estradiol (E(2)) would attenuate the pulmonary vascular structural and hemodynamic changes caused by pulmonary hypertension in utero. To test our hypothesis, we treated chronically instrumented fetal lambs (128 days, term = 147 days) with daily infusions of E(2) (10 microg; E(2) group, n = 6) or saline (control group, n = 5) after partial ligation of the DA. We measured intrauterine pulmonary and systemic artery pressures in both groups throughout the study period. After 8 days, we delivered the study animals by cesarean section to measure their hemodynamic responses to birth-related stimuli. Although pulmonary and systemic arterial pressures were not different in utero, fetal PVR immediately before ventilation was reduced in the E(2)-treated group (2.43 +/- 0.79 vs. 1.48 +/- 0.26 mmHg. ml(-1). min, control vs. E(2), P < 0.05). During the subsequent delivery study, PVR was lower in the E(2)-treated group in response to ventilation with hypoxic gas but was not different between groups with ventilation with 100% O(2). During mechanical ventilation after delivery, arterial partial O(2) pressure was higher in E(2) animals than controls (41 +/- 11 vs. 80 +/- 35 Torr, control vs. E(2), P < 0. 05). Morphometric studies of hypertensive vascular changes revealed that E(2) treatment decreased wall thickness of small pulmonary arteries (59 +/- 1 vs. 48 +/- 1%, control vs. E(2), P < 0.01). We conclude that chronic E(2) treatment in utero attenuates the pulmonary hemodynamic and histological changes caused by DA ligation in fetal lambs.     

Effects of BAY 41-2272, a soluble guanylate cyclase activator, on pulmonary vascular reactivity in the ovine fetus

Nitric oxide (NO)-cGMP signaling plays a critical role during the transition of the pulmonary circulation at birth. BAY 41-2272 is a novel NO-independent direct stimulator of soluble guanylate cyclase that causes vasodilation in systemic and local circulations. However, the hemodynamic effects of BAY 41-2272 have not been studied in the perinatal pulmonary circulation. We hypothesized that BAY 41-2272 causes potent and sustained fetal pulmonary vasodilation. We performed surgery on 14 fetal lambs (125-130 days gestation; term = 147 days) and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic flow transducer was placed on the left pulmonary artery (LPA) to measure blood flow, and a catheter was placed in the LPA for drug infusion. Pulmonary vascular resistance (PVR) was calculated as pulmonary artery pressure minus left atrial pressure divided by LPA blood flow. BAY 41-2272 caused dose-related increases in pulmonary blood flow up to threefold above baseline and reduced PVR by 75% (P < 0.01). Prolonged infusion of BAY 41-2272 caused sustained pulmonary vasodilation throughout the 120-min infusion period. The pulmonary vasodilator effect of BAY 41-2272 was not attenuated by N(omega)-nitro-l-arginine, a NO synthase inhibitor. In addition, compared with sildenafil, a phosphodiesterase 5 inhibitor, the pulmonary vasodilator response to BAY 41-2272 was more prolonged. We conclude that BAY 41-2272 causes potent and sustained fetal pulmonary vasodilation independent of NO release. We speculate that BAY 41-2272 may have therapeutic potential for pulmonary hypertension associated with failure to circulatory adaptation at birth, especially in the setting of impaired NO production.     

Tone-dependent responses to endothelin in the isolated perfused fetal sheep pulmonary circulation in situ

Pulmonary vascular responses to endothelin (ET-1), a peptide derived from endothelial cells in culture, were investigated in the ovine fetus delivered by cesarean section from chloralose-anesthetized ewes with intact umbilical circulation. Circulation to the lower left lobe of the fetal lung was isolated in situ and perfused at constant flow with blood withdrawn from the inferior vena cava. Injection of graded doses of ET-1 into the left pulmonary artery decreased pulmonary arterial perfusion pressure in a dose-related manner. At doses of 100, 300, and 1,000 ng, pulmonary vascular resistance per kilogram body weight (PVR/kg) was decreased 30, 40, and 42%, respectively. However, when fetuses were ventilated with 100% oxygen, 100- and 300-ng doses of ET-1 decreased PVR/kg by 5 and 9%, respectively. In contrast, injection of 1,000 ng of ET-1 resulted in a reversal of the response, and PVR/kg was increased by 70%. Ventilation of the right lung alone resulted in a similar reversal of the vasodilator response to 1,000 ng of ET-1, and a 138% increase in PVR/kg was recorded. These studies demonstrate for the first time that ET-1 has vasodilator activity in the normally high-tone ovine fetal pulmonary circulation. In addition, these results show that ET-1 has vasoconstrictor activity in the newly ventilated low-tone pulmonary vasculature. The present data indicate the pulmonary vascular responses to ET-1 are tone dependent in the ovine fetal pulmonary circulation.     

Distribution of pulmonary vascular pressure as a function of perinatal age in lambs

The distribution of pulmonary vascular resistance (PVR) with respect to compliance was determined using vascular occlusion in isolated lungs from lambs at five ages, from 2 wk before birth to 1 mo of age. The major change in PVR occurred in the pressure gradient across the middle compliant region (delta Pm), which dropped sharply at birth, remained low for 2 wk, and increased at 1 mo. Pulmonary vasoreactivity also varied with ages. Lungs at 0-4 days did not respond to hypoxia and responded poorly to prostaglandin F2 alpha (PGF2 alpha). In contrast, lungs at 13-33 days had significant increases in delta Pm and the gradient across relatively indistensible arterial vessels during hypoxia and increases in all gradients with PGF2 alpha. Ventilation of fetal lungs reduced PVR, mainly because of a 50% reduction in delta Pm. Our results demonstrate that the magnitude and distribution of PVR relative to compliance varied as a function of perinatal age and that pulmonary vasoreactivity depended on postnatal age. The major effect of ventilating fetal lungs was on the middle region.     

Acute pulmonary hemodynamic effects of intravenous copper sulfate: role of alpha-adrenergic system

We investigated the acute pulmonary hemodynamic effects of intravenous copper sulfate (CuSO4) infusion and its mechanism of action in six groups of conscious sheep (total 40). After 300 mg CuSO4 alone, mean pulmonary artery pressure (Ppa) increased from 10.3 to 22.5 Torr and pulmonary artery wedge pressure (Ppaw) from 3.5 to 7.6 Torr, whereas systemic arterial pressure (Psa) increased from 95 to 102 Torr. Cardiac output (Qp) decreased from 4.7 to 3.3 l/min. Pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) increased to 320 and 160% of base line, respectively. The hemodynamic changes correlated well with serum copper, which increased from a base-line value of 0.12 to 3.5 mg/dl after the CuSO4. Serum dopamine beta-hydroxylase increased from 3.2 U/l before CuSO4 injection to 5.7 after its administration, signifying activation of adrenergic nervous system. H1-histamine receptor blockade with chlorpheniramine failed to prevent the effects of CuSO4. Pretreatment with methysergide, a serotonin antagonist, partially attenuated the effects of CuSO4. Phenoxybenzamine, an alpha-adrenergic receptor blocker, and 6-hydroxydopamine, a catecholamine depleting agent, completely blocked the effects of CuSO4. beta-Adrenergic receptor blockade with propranolol enhanced the effects of CuSO4. We conclude, that, in conscious sheep, acute infusion of CuSO4 caused a marked reversible increase in PVR with a slight transient increase in SVR, and this pulmonary hypertension was produced by stimulation of the alpha-adrenergic nervous system.     

Nitric oxide-endothelin-1 interactions after surgically induced acute increases in pulmonary blood flow in intact lambs

Several congenital heart defects require surgery that acutely increases pulmonary blood flow (PBF). This can lead to dynamic alterations in postoperative pulmonary vascular resistance (PVR) and can contribute to morbidity and mortality. Thus the objective of this study was to determine the role of nitric oxide (NO), endothelin (ET)-1, and their interactions in the alterations of PVR after surgically induced increases in PBF. Twenty lambs underwent placement of an aortopulmonary vascular graft. Lambs were instrumented to measure vascular pressures and PBF and studied for 4 h. Before and after shunt opening, lambs received an infusion of saline (n = 9), tezosentan, an ETA- and ETB -receptor antagonist (n = 6), or Nomega-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor (n = 5). In control lambs, shunt opening increased PBF by 117.8% and decreased PVR by 40.7% (P < 0.05) by 15 min, without further changes thereafter. Plasma ET-1 levels increased 17.6% (P < 0.05), and total NOS activity decreased 61.1% (P < 0.05) at 4 h. ET-receptor blockade (tezosentan) prevented the plateau of PBF and PVR, such that PBF was increased and PVR was decreased compared with controls at 3 and 4 h (P < 0.05). These changes were associated with an increase in total NOS activity (+61.4%; P < 0.05) at 4 h. NOS inhibition (L-NNA) after shunt placement prevented the sustained decrease in PVR seen in control lambs. In these lambs, PVR decreased by 15 min (P < 0.05) but returned to baseline by 2 h. Together, these data suggest that surgically induced increases in PBF are limited by vasoconstriction, at least in part by an ET-receptor-mediated decrease in lung NOS activity. Thus NO appears to be important in maintaining a reduction in PVR after acutely increased PBF.