Vascular potassium channels mediate oxygen-induced pulmonary vasodilation in fetal lambs

The pulmonary vascular resistance decreases at birth secondary to release of endothelium-derived nitric oxide (EDNO). EDNO release is a calcium-dependent process, and endothelial potassium (K+) channels regulate intracellular calcium flux. We investigated the hypothesis that potassium channels mediate oxygen-induced pulmonary vasodilation and EDNO release in fetal lambs. We instrumented 18 near-term fetal lambs at 122-126 days of gestation to measure pulmonary pressures, flow, and resistance. We studied hemodynamic effects of (1) 100% oxygen; (2) pinacidil, an ATP-sensitive K+ (KATP) channel agonist, and (3) S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. We studied the effects of glybenclamide, a K(ATP) channel antagonist, tetraethylammonium chloride (TEA), a preferential KCa channel antagonist, and nitro-L-arginine (NLA), an NO synthase inhibitor, on the response to some of the above agents. Oxygen-induced pulmonary vasodilation was inhibited by both glybenclamide and TEA, indicating that K(ATP) and K(Ca) channels mediate pulmonary vasodilator response to oxygen. Blocking NO synthesis with NLA inhibited pinacidil-mediated pulmonary vasodilation, indicating that K(ATP) channel activation stimulates NO release. SNAP-mediated pulmonary vasodilation was inhibited by TEA, but not glybenclamide, indicating that K(Ca) channels, but not K(ATP) channels, mediate effects of NO on vascular smooth muscle relaxation. In conclusion, K+ channels mediate oxygen-induced pulmonary vasodilation in fetal lambs. K(ATP) channels appear to mediate EDNO release, while K(Ca) channels probably mediate NO effects on vascular smooth muscle.     

Adenosine and ATP cause nitric oxide-dependent pulmonary vasodilation in fetal lambs

We investigated the hypothesis that the purine nucleotide ATP and its nucleoside adenosine cause pulmonary vasodilation in fetal lambs by the release of nitric oxide (NO). We also investigated the potential role of K(+)(ATP) channels in mediating the effects of ATP and adenosine on NO. We surgically prepared 28 fetal lambs to measure pulmonary and systemic pressures and pulmonary flow. We investigated the effects of glibenclamide and pinacidil (inhibitor and agonist, respectively, for K(+)(ATP) channels), N-nitro-L-arginine (N-LA) and its methyl ester, N-nitro-L-arginine methyl ester (L-NAME) (inhibitors of endothelium-derived NO synthesis), and U46619 (a thromboxane mimetic) on pulmonary vasodilation caused by adenosine and ATP. Adenosine decreased the pulmonary artery pressure and pulmonary vascular resistance (PVR) at doses of 0.08-2.5 microM/kg/min and increased the left pulmonary flow at doses of 0.3-2.5 microM/kg/min in control experiments. N-LA, L-NAME and glibenclamide attenuated the effects of adenosine at doses of < 2.5 microM/ kg/min and pinacidil potentiated its effects. ATP decreased the pulmonary artery pressure and PVR and increased the pulmonary flow at doses of 0.15-2.5 microM/kg/min in control experiments. N-LA and L-NAME attenuated the effects of ATP at doses of < 2.5 microM/kg/min, whereas glibenclamide and pinacidil had no effect on the response to ATP. U46619 increased the basal pulmonary vascular tone, but did not significantly alter the vasodilative responses to ATP and adenosine. In conclusion, adenosine and ATP cause NO-dependent pulmonary vasodilation in fetal lambs. The activation of K(+)(ATP) channels plays a role in adenosine-induced pulmonary vasodilation. The mechanism by which ATP causes NO release and pulmonary vasodilation requires further investigation.     

Betamethasone attenuates oxidant stress in endothelial cells from fetal lambs with persistent pulmonary hypertension

We investigated the effects of betamethasone on oxidative stress and impaired vasodilation in a lamb model of persistent pulmonary hypertension (PPHN). We treated pregnant ewes following fetal ductal ligation with betamethasone or saline for 48 h before delivery. Response of fetal pulmonary arteries to nitric oxide synthase (NOS) agonist adenosine triphosphate (ATP) and nitric oxide (NO) donor, s-nitroso-n-acetyl-penicillamine (SNAP) was determined in tissue bath. Pulmonary artery endothelial cells (PAEC) from fetal lambs with ductal ligation or sham ligation were treated with betamethasone or its vehicle for 48 h. Expression of endothelial NOS (eNOS), endothelin, endothelin-B (ET-B) receptor, and CuZn- and Mn-superoxide dismutase (SOD) in PAEC was studied. Intracellular cGMP and superoxide levels and interaction of eNOS with heat shock protein 90 (Hsp90) were determined in PAEC. Antenatal betamethasone improved the relaxation response of pulmonary arteries to ATP and SNAP in PPHN. PPHN was associated with decreases in eNOS and ET-B receptor and increase in prepro-endothelin mRNA levels. Betamethasone decreased prepro-endothelin mRNA and ET-1 pro-peptide levels and increased eNOS and MnSOD protein levels in PPHN. Betamethasone reversed the increased superoxide/decreased cGMP levels and restored Hsp90-eNOS interactions in PPHN. Betamethasone reduces oxidative stress and improves response of pulmonary arteries to vasodilators in lambs with PPHN.     

Alkalosis attenuates hypoxic pulmonary vasoconstriction in neonatal lambs

Hyperventilation (respiratory alkalosis) is an important treatment for persistent pulmonary hypertension in neonates. The precise way that hyperventilation attenuates hypoxic pulmonary vasoconstriction is unclear. We studied the effect of alkalosis on hypoxia-induced pulmonary vasoconstriction in 13 acutely instrumented, pentobarbital anesthetized, neonatal lambs. We specifically examined the relative effects of a metabolic alkalosis versus a respiratory alkalosis on hypoxic pulmonary vasoconstriction and compared these results to the control response to hypoxia without alkalosis. Hypoxic pulmonary vasoconstriction was significantly milder whenever the animal was alkalotic, regardless of whether the alkalosis was respiratory of metabolic. Thus, the elevated pHa rather than decreased PaCO2 during hyperventilation appears to be the major factor in moderating the response of the pulmonary vessels to acute hypoxia in this neonatal lamb model.     

Tezosentan, a combined parenteral endothelin receptor antagonist, produces pulmonary vasodilation in lambs with acute and chronic pulmonary hypertension.

OBJECTIVE: To investigate the hemodynamic effects of tezosentan in the intact lamb both at rest and during acute and chronic pulmonary hypertension. DESIGN: Prospective, randomized experimental study. SETTING: University-based research laboratory. SUBJECTS: Lambs with and without pulmonary hypertension. INTERVENTIONS: Six newborn lambs were instrumented to measure vascular pressures and left pulmonary blood flow. The hemodynamic effects of tezosentan (0.5, 1.0, 5.0 mg/kg, intravenously) were studied at rest and during U46619-induced pulmonary hypertension. Following in utero placement of an aortopulmonary vascular graft, nine additional lambs with increased pulmonary blood flow and chronic pulmonary hypertension (shunt) were also studied at 1 wk (n = 5) and 8 wks (n = 4) of age. MEASUREMENTS AND MAIN RESULTS: At rest, tezosentan had no significant effect on any of the variables. During acute U46619-induced pulmonary hypertension, tezosentan caused a dose-dependent decrease in pulmonary arterial pressure (from 5.9% +/- 4.7 to 16.0% +/- 10.7; p < .05) and pulmonary vascular resistance (from 6.2% +/- 8.0 to 21% +/- 8.8; p < .05). Mean systemic arterial pressure was unchanged. In 1- and 8-wk-old shunt lambs with increased pulmonary blood flow, tezosentan (1 mg/kg) produced potent nonselective pulmonary vasodilation. CONCLUSIONS: Tezosentan, a combined endothelin receptor antagonist optimized for parenteral use, induces potent selective pulmonary vasodilation during acute U46619-induced pulmonary hypertension and potent nonselective vasodilation in chronic pulmonary hypertension secondary to increased pulmonary blood flow. In general, the hemodynamic effects of bolus doses of tezosentan occurred within 60 secs of administration and lasted approximately 5-10 mins. The hemodynamic profile of intravenous tezosentan may make it a useful adjunct therapy for acute pulmonary hypertensive disorders and warrants further study.     

Endothelin-A receptor blockade in porcine pulmonary hypertension

Endothelin-1 can cause pulmonary vasoconstriction via endothelin-A (ET(A)) receptor activation. We hypothesized that ET(A) blockers (EMD 122946 and BQ 610) would reduce hypoxia-induced (HYP) but not group B streptococcal infusion (GBS)-induced pulmonary hypertension in a juvenile whole animal model. Pulmonary hypertension was created by exposing chronically instrumented piglets to HYP (n = 12) or heat-killed GBS (n = 11). ET(A) blockade was produced by increasing bolus doses of EMD122946 or BQ 610. Pulmonary arterial pressure (PAP), systemic arterial pressure (SAP), left atrial pressure, central venous pressure, and cardiac output were continuously measured. Pulmonary and systemic vascular resistance indices (PVRI and SVRI) were calculated. HYP doubled PAP and PVRI. Both ET(A) blockers decreased PAP and PVRI in a dose-dependent manner in HYP, with high doses decreasing PVRI to baseline and reducing PAP by 50%. GBS also doubled both PAP and PVRI. EMD 122946 did not change PAP or PVRI in GBS, although BQ 610 markedly increased PVRI (>100% increase with 0.15 mg/kg) and showed a trend toward increasing PAP. Both models showed minimal (<25%) changes in SAP or SVRI. Neither ETA blocker changed baseline hemodynamics in the absence of HYP or GBS. PaO(2) did not change with GBS but decreased with BQ 610. ET(A) receptor blockade attenuated hypoxic, but not GBS induced pulmonary hypertension. BQ 610 worsened PVRI and oxygenation in the GBS model. Differences in response to ET(A) blockade in pulmonary hypertension may be seen depending on the etiology (hypoxia versus infection-associated), and the specific ET(A) antagonist used.     

Effect of beta-adrenergic receptor blockade on responses to acute hypoxemia in lambs

We studied the effects of beta-adrenergic receptor blockade on general circulatory and metabolic responses to moderate (FIO2 = 0.09) acute hypoxemia in newborn (protocol 1) and 3-wk-old (protocol 2) lambs, and on regional blood flow distribution in newborn lambs (protocol 1). Via a left thoracotomy we placed an electromagnetic flow transducer around the ascending aorta and inserted various vascular catheters. After 2 days of recovery, the lambs were studied. In protocol 1, we measured cardiovascular variables and regional blood flow distribution during control conditions, after 45 min of acute hypoxemia, and after 0.5 mg/kg of propranolol during acute hypoxemia. In protocol 2, we measured cardiovascular variables during control conditions and after 45 min of acute hypoxemia with and without propranolol pretreatment. In both groups, propranolol limited the increase in cardiac output and heart rate caused by hypoxemia, and thus decreased oxygen delivery. However, propranolol also decreased oxygen consumption so that pulmonary arterial pO2 was either higher (protocol 1) or the same (protocol 2) as during acute hypoxemia alone. Neither metabolic acidosis nor hypothermia ensued. In protocol 1, propranolol decreased renal, carcass, and most importantly, myocardial blood flows. However, myocardial O2 consumption also fell, coronary sinus pO2 increased, and blood was redistributed toward the subendocardium, suggesting that myocardial perfusion improved. Thus, beta-adrenergic receptor blockade during acute moderate hypoxemia may have a beneficial effect by reducing total body and myocardial oxygen demand in excess of the reduction in oxygen delivery.     

Thromboxane is not responsible for the high pulmonary vascular resistance in fetal lambs

The factors responsible for the high pulmonary vascular resistance in the fetus are not well known. Thromboxane (TX) A2 is a potent pulmonary vasoconstrictor. To determine whether TXA2 may play a role in fetal pulmonary vasoconstriction, we infused the specific TX synthetase inhibitor U63,557A into eight chronically instrumented fetal lambs (134-137 days gestational age, full term 145 days) and measured pulmonary blood flow, pulmonary and systemic arterial pressure, and heart rate. U63,557A (3 mg/kg as a bolus then 3 micrograms/kg/min for 120 min infused in the main pulmonary artery) did not change pulmonary blood flow, pulmonary mean arterial pressure, and pulmonary vascular resistance during the infusion and during 2 h following the end of the infusion. During the infusion, TXB2 arterial plasma concentrations decreased from 106.1 +/- 17.5 to 8.7 +/- 2.9 pg/ml. In three of the fetal lambs, immediately after the 2-h infusion of U63,557A, we infused the leukotriene end-organ antagonist FPL 57231 into the main pulmonary artery (1 mg/kg/min for 60 min). TXA2 synthesis inhibition did not prevent the pulmonary vasodilation induced by FPL 57231. Pulmonary blood flow increased from 64.8 +/- 24.4 to 669.5 +/- 65.6 ml/min/100 g lung tissue during the FPL 57231 infusion. We conclude that TXA2 does not play a role in the maintenance of elevated fetal pulmonary vascular tone, either directly or as a mediator of leukotriene action.     

The effect of alpha-adrenergic blockade on the pulmonary vascular response to dopamine in neonatal lambs

The effect of alpha-adrenergic blockade by phentolamine on the pulmonary vascular response to dopamine was studied in chronically prepared newborn lambs. Dopamine was administered at doses of 2.7 micrograms . kg-1 . min-1, 27 micrograms . kg-1 . min-1 and 270 micrograms . kg-1 . min-1 with and without alpha-adrenergic blockade. Dopamine infusion at 270 micrograms . kg-1 . min-1 caused a rise in the mean pulmonary artery pressure from 22 +/- 3.2 mmHg (mean +/- S.E.) at baseline to 36 +/- 4.1 mmHg (P less than 0.001). This rise was unaffected by alpha-adrenergic blockade. Dopamine infusion alone did not change pulmonary blood flow, but, in the presence of alpha-adrenergic blockade, pulmonary blood flow rose from 190 +/- 12 ml . min-1 . kg-1 at baseline to 280 +/- 13 ml . min-1 . kg-1 at the maximum dopamine infusion rate (P less than 0.001). Pulmonary vascular resistance was the same before and after alpha-adrenergic blockade and did not change from the baseline value during dopamine infusion.     

Piriprost: a putative leukotriene synthesis inhibitor increases pulmonary blood flow in fetal lambs

Leukotrienes may control fetal pulmonary vascular tone since infusions of putative leukotriene receptor antagonists markedly increase pulmonary blood flow and decrease pulmonary vascular resistance in fetal lambs. This hypothesis would be strengthened if inhibition of leukotriene synthesis also produced similar hemodynamic changes. We therefore studied the effects of piriprost (U 60257), a putative leukotriene synthesis inhibitor, on thirteen fetal lambs at 137 to 140 days gestation. In preliminary studies in four fetal lambs, doses of U 60257 greater than 20 mg/kg increased pulmonary blood flow. In the nine other fetal lambs, U 60257 (31.7 +/- 4.1 mg/kg) increased pulmonary blood flow by 502% (p less than 0.05) and decreased pulmonary vascular resistance by 87% (p less than 0.05). Pulmonary arterial and left atrial pressures were unchanged. Descending aortic pressure was increased (p less than 0.05) and heart rate was decreased (p less than 0.05). The abilities of both putative leukotriene synthesis inhibitors and leukotriene receptor antagonists to similarly increase fetal pulmonary blood flow and decrease pulmonary vascular resistance are consistent with the hypothesis that leukotrienes play a role in regulating fetal pulmonary vascular tone.     

Increased arterial pH, not decreased PaCO2, attenuates hypoxia-induced pulmonary vasoconstriction in newborn lambs

Mechanically induced hyperventilation is used in the treatment of newborn infants with persistent pulmonary hypertension syndrome to induce respiratory alkalosis, which may attenuate their pulmonary vasoconstriction. Whether this treatment is effective because of the increase in arterial pH or the decrease in Paco2 was investigated in nine sedated, mechanically ventilated newborn lambs with hypoxia-induced pulmonary vasoconstriction. We found that respiratory alkalosis and metabolic alkalosis were equally effective in attenuating hypoxia-induced pulmonary vasoconstriction, but that hypocapnia (low Paco2 with a normal arterial pH) was ineffective. These results indicate that increased arterial pH, not decreased Paco2, attenuates hypoxia-induced pulmonary vasoconstriction in newborn lambs and possibly the pulmonary vasoconstriction in newborn infants with persistent pulmonary hypertension syndrome.     

Compensatory renal hypertrophy in fetal lambs

Compensatory renal hypertrophy was studied in fetal lambs during midgestation. Functional adaptation was correlated with anatomica and biochemical changes by measuring glomerular filtration and clearance of para-amino hippurate (PAH). Normal intrauterine body growth and kidney growth by changes in RNA and DNA over a 72-hr period were studied in twin fetuses. Seventy-two hr after left uninephrectomy in single fetuses, there was a significant increase in weight of the renoprival right kidney as well as a significant increase in renal cortical content of RNA and DNA. The rate of increase in RNA was greater than the increase in DNA. Preliminary studies suggest that an increase in renal function parallels renal hypertrophy in fetal lambs.     

Indomethacin prevents ventilation-induced decreases in pulmonary vascular resistance of the middle region in fetal lambs

Previously, we report that the major site of pulmonary vascular resistance in fetal lambs occurred in the middle region defined by vascular occlusion, and that this region exhibited the greatest decrease upon ventilation with O2. To assess the relative individual contributions of ventilation and oxygenation to this decrease, we determined the distribution of pressures across the pulmonary circulation in isolated perfused lungs from 20 fetal lambs (131 - 137 d gestation) by inflow and outflow vascular occlusions. A membrane oxygenator was included in the extracorporeal circuit to control the PO2 at 4 kPa (30 torr) in the unventilated fetal lungs. Half of the fetal lungs were ventilated first without changing the initial gas tensions, and the others were oxygenated first by changing the initial gas tension to a hyperoxic mixture [PO2 = 26.6 kPa (200 torr)] without ventilation. Finally, both groups of lungs were ventilated and oxygenated. In addition, indomethacin was added to the perfusate (0.112 mM, or 40 micrograms/mL) in half of the preparations in each group to determine the effect of prostaglandins on the distribution of pressures during these conditions. The decrease in the total pulmonary vascular resistance with ventilation and/or oxygenation was primarily due to changes in the middle pressure gradient (delta Pm). In fetal lungs without indomethacin, ventilation without oxygenation reduced delta Pm from 6.1 +/- 0.8 to 2.5 +/- 1.0 kPa, or 74% of the total ventilation- and oxygenation-induced decrease in delta Pm (final value = 1.2 +/- 0.6 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypotension in fetal and newborn lambs; different patterns of reflex heart rate control revealed by autonomic blockade

Reflex heart rate (HR) responses to hypotension were studied in chronically instrumented fetal and newborn lambs. Studies spanned 106-141 days of gestation and 1-22 days after birth. Brief hypotensive stimuli (10 s) were produced by inflation of a cuff implanted around the inferior vena cava; HR and mean arterial pressure (MAP) were recorded from a carotid arterial catheter. Autonomic nervous control of HR was examined using selective sympathetic beta-adrenergic blockade (propranolol 1 mg/kg), cholinergic blockade (atropine 0.2-0.3 mg/kg), and total autonomic blockade (propranolol plus atropine). In newborn lambs (n = 4) HR increased progressively as MAP was reduced in the range 5-50%. Tachycardia during mild hypotension (less than 15% MAP fall) was due to sympathetic activation as it was abolished by propranolol. During severe hypotension (greater than 30% MAP fall) tachycardia was reduced by selective beta-adrenergic blockade and by cholinergic blockade, and totally abolished by total autonomic blockade; thus withdrawal of vagal tone plus augmentation of sympathetic activity contribute to the increase of HR in response to large MAP falls in the newborn. Fetal lambs (n = 4) responded with tachycardia in mild hypotension (less than 15% MAP fall) but this was reversed when hypotension was severe (greater than 30% MAP fall). The primary tachycardia was due to sympathetic activation and was indistinguishable from the newborn response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal phosphate clearance in fetal lambs.

The purpose of this study was to investigate the possible role of diminished phosphate clearance by the fetal kidney in production of relative fetal hyperphosphatemia. Stimuli known to affect renal phosphate clearance in adults were investigated in young fetal lambs. Our studies confirm that the fetal lamb kidney responds to exogenous and endogenous parathyroid hormone (PTH) with inhibition of tubular phosphate reabsorption. Renal tubular phosphate reabsorption in the fetus is in part related to sodium reabsorption. These studies indicate that so-called "immaturity" of renal phosphate clearance in utero is not a significant factor in production of fetal hyperphosphatemia.     

Ontogeny of neutrophil chemotaxis in fetal lambs

Ontogeny of neutrophil chemotactic response using endotoxin activated adult sheep plasma as a source of complement derived chemotactic factor was examined in fetal lambs of gestational age 120-150 days. (Gestational period in sheep is approximately 150 days.) Neutrophils from fetal lambs of gestational age 120-130 days failed to respond to this chemotactic factor whereas neutrophils from fetal lambs above 131 days of gestational age responded at levels comparable to adult values. Examination of neutrophil chemotaxis in older fetuses using a different chemotactic factor derived from mitogen stimulated adult mononuclear cells revealed a selective failure of fetal neutrophils to respond to lymphocyte-derived chemotactic factor in the presence of a normal response to complement derived chemotactic factor. Among prematurely delivered twin fetuses alterations in comparison with the first twin or age-matched controls in peripheral neutrophil count (increase) and in chemotaxis (increase or decrease) were noted in second of twins delivered greater than or equal to 20 min after the first lamb, suggesting an extreme sensitivity of neutrophil functions to a variety of influences, similar to that seen in humans.     

Inhaled nitric oxide attenuates hyperoxic lung injury in lambs

Cytochrome P450 (CYP) inhibition with cimetidine reduces hyperoxic lung injury in young lambs. Nitric oxide (NO), also a CYP inhibitor, has been shown to either aggravate or protect against oxidant stress depending on experimental context. The objective of this study was to determine whether NO, like cimetidine, would protect young lambs against hyperoxic lung injury, and whether its effect was associated with CYP inhibition. Three groups of lambs were studied: 1) room air exposure, 2) >95% O2, and 3) >95% O2 plus inhaled NO. After 72 h, hyperoxia alone resulted in a significant increase in arterial P(CO2) and number of polymorphonuclear leukocytes in bronchoalveolar lavage (BAL), and a significant decrease in arterial/alveolar O2 tension (a/A). The addition of inhaled NO significantly decreased the hypercarbia and BAL polymorphonuclear cellular response associated with hyperoxia but had no beneficial effect on a/A ratio. There were no significant differences in F2-isoprostanes or isofurans (markers of lipid peroxidation) measured in BAL or lung tissue among study groups. No intergroup differences were detected in BAL epoxyeicosatrienoic acid levels (index of CYP activity). The results of this study indicate that hypercarbia and inflammation accompanying hyperoxic lung injury in young lambs can be attenuated by inhaled NO. However, this study provides no direct evidence that NO is inhibiting CYP-mediated oxidant lung injury.