Ann Thorac Surg 2000;70:1522-1530
© 2000 The Society of Thoracic Surgeons
Original articles: cardiovascular
Characterization of the pulmonary arterial response to endothelin-1 and bosentan in neonatal pigs
Patrick W. Domkowski, MD, PhDa,
John T. Cockerham, MDa,
Peter A. Kot, MDa,
Jeff L. Myers, MD, PhDa,
Richard A. Hopkins, MDa
a Department of Surgery, Brown University School of Medicine, Providence, Rhode Island, USA
Address reprint requests to Dr Hopkins, Cardiothoracic Surgery, 164 Summit Ave, Providence, RI 02906
e-mail: rahopkins{at}lifespan.org
Presented at the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
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Abstract
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Background. This study determined the pulmonary vascular responses to intravenous (IV) administration of endothelin-1 (ET-1) before and after an IV bolus of bosentan (Ro 47-0203), an endothelin receptor antagonist, in anesthetized open-chest 48-hour-old and 2-week-old Yorkshire pigs.
Methods. Eighteen 48-hour-old and 25 2-week-old pigs were randomly allocated to receive either (1) 400 ng · kg-1 · min-1 of ET-1 or (2) 5 mg/kg or 10 mg/kg of Ro 47-0203 followed by 400 ng · kg-1 · min-1 of ET-1 over a 10-minute interval. Pulmonary vascular resistance (PVR, dyne sec/cm-5), elastic modulus (EYo, dyne/cm2), and characteristic impedance (Zo) were determined (± SEM).
Results. In 48-hour-old pigs, ET-1 decreased pulmonary artery pressure (PAP, dyne/cm2; 21,317 ± 1833 versus 17,757 ± 1823; p = 0.003). In 2-week-old pigs, ET-1 elevated PAP (19,009 ± 1834 versus 21,935 ± 2104; p = 0.003) and PVR (1624 ± 254 versus 2302 ± 416; p = 0.001), whereas bosentan abolished the ET-1 induced pulmonary and systemic vasoconstriction. Neither agent altered EY or Zo.
Conclusions. ET-1 caused a pulmonary depressor response in 48-hour-old pigs and a constrictor response in 2-week-old pigs, whereas bosentan inhibited the ET-1 induced pulmonary arteriolar vasoconstriction in 2-week-old pigs. The response to ET-1 changes from dilation in 48-hour-old pigs (neonates) to constriction in 2-week-old pigs (infants) suggests a maturational dependent alteration in ET receptors during the first 2 weeks of life. These data suggest that bosentan may have potential clinical application in the treatment of newborn pulmonary hypertensive episodes as it ablated ET-1 induced pulmonary vasoconstriction, while maintaining systemic pressure.
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Introduction
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Endothelin-1 (ET-1) a potent vasoactive peptide, was isolated by Yanagisawa and colleagues [1] from porcine aortic endothelial cells. It is one of three similar but genetically distinct isoforms (ET-1, ET-2, and ET-3) known to occur endogenously in most mammals [2]. In the newborn pig pulmonary circulation ET-1 possesses both vasodilator and vasoconstrictor activity [3–6]. Studies have demonstrated that the vasodilator response produced by ET-1 in the newborn pig pulmonary circulation is mediated in part by both the activation K+ channels and the release of EDRF [7]. In the fully mature pig, ET-1 consistently induces a potent and prolonged pulmonary vasoconstriction [8–10]. During early maturation the porcine pulmonary vascular response changes from dilation to constriction. It has been hypothesized that there may be a receptor density change within the pulmonary circulation during maturation [10, 11]. The exact time that the pulmonary circulation alters its response to ET-1 remains unclear.
Although four distinct endothelin receptors have been cloned—ETA, ETB, ETC, and ETAX—ETA and ETB receptors appear to predominate in mammalian tissue [11]. In addition, little information is available on the hemodynamic or elasticity response to blockade of these receptors in the intact newborn pig pulmonary circulation. An endothelin receptor antagonist, bosentan (Ro 47-0203) is a potent competitive inhibitor of both ETA and ETB receptors. Unlike most previously reported ET-1 antagonists, bosentan is a nonpeptide and is therefore both orally and parenterally active [12, 13]. The effects of ET-1 and Ro 47-0203 on the mean pulmonary pressure/flow relationship as reflected by alterations in pulmonary vascular resistance have been characterized in adult animals [4, 5]. However, the stress-strain relationship and the pulsatile pressure/flow response to any of these agents as quantified by the elastic modulus and hydraulic impedance, respectively, have not been determined. Moreover, the relationship between Ro 47-0203 and the ET-1 response elicited in the intact maturing newborn pig pulmonary circulation is unknown.
Thus the purpose of this study was twofold. The first objective was to determine the acute pulmonary hemodynamic effects of an intravenous infusion of porcine ET-1 both before and after infusion of Ro 47-0203 in 48-hour-old and 2-week-old anesthetized, open-chest Yorkshire pigs. The second objective was to determine the pulmonary vascular impedance and the elastic modulus response to porcine ET-1 both before and after intravenous infusion of Ro 47-0203 in these same animals.
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Material and methods
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Surgical preparation
Eighteen, 48-hour-old (± 4 hours, mean weight = 1.5 to 2.8 kg, mean body surface area = 0.15 ± 0.1 m2) and 25 2-week-old (mean weight = 4.0 to 5.0 kg, mean body surface area = 0.21 ± 0.1 m2) Yorkshire pigs of either sex were anesthetized with intravenous thiopental sodium (25 mg/kg). A half dose of this anesthetic agent was given every 20 minutes to maintain an adequate level of anesthesia. Additional anesthetic was administered when the heart rate was greater than 120 beats per minute or the mean systemic arterial pressure was greater than 70 mm Hg. All animals underwent endotracheal intubation and were placed in the supine position. The ear vein was catheterized and sufficient pancuronium bromide (0.1 mg/kg intravenously) was administered to produce complete muscle relaxation. The animals were mechanically ventilated, initially with a fraction of inspired oxygen (FIO2) of 1.0 with a pediatric positive pressure ventilator (Health dyne 105, Marietta, GA). Positive inspiratory pressure was preset between 20 and 30 cm H2O and respiratory rate between 9 and 10 ventilations per minute. These ventilatory settings achieve a partial pressure of PaCO2 between 30 and 40 mm Hg and had no effect on baseline hemodynamic measurements. To avoid the effects of respiratory motion on pulmonary artery pressure and flow, ventilation was briefly interrupted during data collection intervals without any observable changes in pulmonary or systemic parameters [14]. A positive end-expiratory pressure of 3 cm H2O was maintained intraoperatively to prevent atelectasis.
The surgical technique and instrumentation of the animals have previously been described in detail [14, 15]. Briefly, following a median sternotomy, the pericardium was incised longitudinally and two 4-0 silk (Ethicon, Somerville, NJ) pericardial purse strings were placed to provide exposure of the heart and great vessels. The main pulmonary artery was dissected free from the aorta. To guarantee complete separation of the pulmonary and systemic circulations in 48-hour-old and 2-week-old pigs, a medium-sized titanium clip (Ethicon, Rochester, NY) was used to occlude the ductus arteriosus. High-fidelity ultra light weight (10 mg) sonomicrometer piezo electric crystals were attached via a 4-0 silk stitch placed on the outside lateral wall of each side of the main pulmonary artery. The crystals were positioned 180 degrees from each other to achieve an accurate measurement of pulmonary artery diameter. Proximal to the crystals (toward the heart) an appropriately sized ultrasonic flow probe (Transonic Systems, Ithaca, NY) was carefully fitted around the main pulmonary artery to ensure good electrical contact of the vessel while avoiding significant constriction. A high-fidelity Millar pressure transducer (model MPC-500; Millar Inc, Houston, TX) was passed into the left atrial appendage and a second high-fidelity Millar pressure transducer (model SPC-320) was inserted into the main pulmonary artery. Premeasurement of the catheter length in relation to the main pulmonary artery ensured that the transducer tip was positioned beyond the ultrasonic flow probe. A third high-fidelity pressure transducer was inserted into the right internal carotid artery for accurate measurement of systemic blood pressure.
Data collection and analysis
The impedance analysis has been previously described [14, 15]. Our elastic modulus (EY) was derived from tension-strain curves delineated in the Appendix.
Instrument calibration
High-fidelity pressure transducers were calibrated before and after each experiment. Data were disregarded if there was ±5% drift in calibration measurements from baseline. Cross correlation (using Fourier analysis in both frequency and time domains) synthetic electronic input signals of digitized waveforms determined less than a 0.05% phase shift between channels, and therefore no internal phase correction was required.
Calibration of the flow probes has been previously described in detail [14]. The transonic flow meter system is linear to 60 Hz with a flat amplitude response to 35 Hz [16]. All mathematical derivations and formulas are delineated in Appendix A. Calibration of the piezoelectric crystals was performed on a synthetic elastic tube of known diameter. Analog measurements were verified to be within 1% of the tube diameter. Electrical crystal measurements greater than 1% away from the tube diameter were discarded. Phase lags were also independently verified for pressure, flow, and diameter signals generated from each respective amplifier.
Experimental protocol
Human/porcine endothelin-1 (Sigma Chemical Co, St. Louis, MO) was solubilized in 0.9% sterile NaCl for intravenous infusion. Bosentan was solubilized in a 1:1 equal volume to volume ratio of 0.9% sterile NaCl and sterile deionized water. All drugs were administered through the right internal jugular vein. Forty-eight-hour and 2-week-old pigs were randomly allocated to receive either a 10-minute intravenous infusion of 400 ng · kg-1 · min-1 of ET-1, or an intravenous bolus of either 5 mg/kg or 10 mg/kg of Ro 47-0203 followed by a 10-minute infusion of 400 ng · kg-1 · min-1 of ET-1. Acute hemodynamic measurements for ET-1 were obtained at baseline, at three separate times (2 minutes, 6 minutes, and 10 minutes) during delivery of each drug, and 10 minutes following cessation of drug administration. Hemodynamic measurements for Ro 47-0203 were obtained at 1 and 3 minutes after the bolus injection and averaged because there were no significant differences between these two sets of measurements. Based on dose responses reported in other hemodynamic studies, a submaximal dose of ET-1 was selected for use throughout this investigation [5, 17].
All experiments were preapproved by the Animal Care and Use Committee. All animals received humane care in compliance with the "Principles of Laboratory Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Institutes of Health (NIH publication No. 80-23, revised 1985).
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Results
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Endothelin-1, 48-hour-old pigs
Standard hemodynamics
Mean pulmonary artery pressure (PAP) decreased significantly (p = 0.003) during ET-1 infusion in the 8 pigs (Table 1). Pulmonary vascular resistance (PVR) and pulmonary artery flow (PAF) both tended to decrease during ET-1 infusion; however, the overall changes were not significant for either parameter (Table 1). The pH (7.35 to 7.38) and PaCO2 (35 to 40 mm Hg) both remained within normal range during the duration of each experiment. The PaCO2 ranged between 450 and 530 mm Hg, which is consistent when ventilated on an FIO2 of 1.0. Mean left atrial pressures (LAP) were not significantly changed during the ET-1 experiments. Aortic pressure (AP) (60,590 ± 6,386 dyne/cm2) increased significantly during (75,067 ± 8,081 dyne/cm2, p = 0.045) and for 10 minutes after (72,586 ± 8,495 dyne/cm2, p = 0.045) ET-1 infusion.
Pulsatile data
The baseline impedance modulus indicated a first minimum at 2 Hz with the phase angle becoming positive between 6 and 7 Hz. During ET-1 infusion the first minimum was shifted to the right, while the phase angle became positive between 3 and 4 Hz (Fig 1A). Input mean impedance (Zm) was not altered by ET-1 during or after ET-1 administration (Table 1). Although characteristic impedances (Zo2-7 Hz and Z3-10 Hz) were not altered, a substantial decrease in the oscillations of the impedance moduli were noted throughout the higher frequencies (Fig 1A). The elastic modulus was not significantly changed from baseline during or after endothelin-1 infusion.
Endothelin-1, 2-week-old pigs
Standard hemodynamics
ET-1 infusion increased PAP (p = 0.001) and PVR (p = 0.05) significantly in these 10 pigs. These parameters remained elevated for 10 minutes after infusion of ET-1 (Table 1). ET-1 tended to decrease PAF; however, the change was not significant. The pH (7.35 to 7.38) and PaCO2 (35 to 40 mm Hg) remained within the normal range throughout the experiment. Mean LAP was not significantly changed during these experiments. Baseline aortic pressure (59,856 ± 5,231 dyne/cm2) increased significantly during ET-1 infusion (86,193 ± 5,183 dyne/cm2, p = 0.013) and remained elevated during the 10 minutes it was monitored after drug infusion (90,758 ± 4,482 dyne/cm2, p = 0.001).
Pulsatile data
The baseline impedance modulus reached a first minimum between 1 and 2 Hz with the phase angle becoming positive for the first time between 3 and 4 Hz. During ET-1 administration there were no significant changes in the occurrence of the frequency of the first minimum (Fig 1B). Zm was augmented (p < 0.05) during and after ET-1 infusion (Table 1). Characteristic impedances (Zo2-7 Hz and Z3-10 Hz) were not altered during ET-1 infusion. The elastic modulus was not significantly changed from baseline during or after infusion of ET-1 (Table 1).
Bosentan (Ro 47-0203)/endothelin-1, 48-hour-old pigs
Standard hemodynamics
Baseline PAP was increased (p = 0.017) following 5 mg/kg administration of Ro 47-0203 (Table 2) to 10 pigs. Baseline PVR tended to increase; however, it did not reach significance (Table 2). PAP remained elevated with subsequent infusion of ET-1. The pH (7.35 to 7.39) and PaCO2 (35 to 39 mm Hg) both remained within normal range during the duration of each experiment. The PaCO2 ranged between 400 and 510 mm Hg, which is consistent when ventilated on an FIO2 of 1.0. LAP was not altered during Ro 47-0203 administration (5 mg/kg) or subsequent ET-1 administration. Baseline AP (57,704 ± 7,958 dyne/cm2) was not significantly altered after 5 mg/kg bolus administration of bosentan (56,017 ± 8,731 dyne/cm2) or during subsequent infusion of ET-1 (61,823 ± 8,590 dyne/cm2).
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Table 2. Pulmonary Vascular Responses to Pretreatment With Bosentan (Ro 47-0203 at 5 mg/kg and 10 mg/kg) Followed by Subsequent Endothelin-1 Infusion in 48-Hour Pigs
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Administration of 10 mg/kg of Ro 47-0203 caused a significant increase in baseline pulmonary artery pressure (p = 0.02, Table 2). Baseline PAF and PVR were not significantly altered after Ro 47-0203 (10 mg/kg) administration (Table 2). Subsequent infusion of ET-1 caused a further increase in PAP; however, PVR was not significantly altered. LAP was not significantly altered during 10 mg/kg of Ro 47-0203 infusion or during subsequent ET-1 infusion. Baseline AP (66,218 ± 5,549 dyne/cm2) was not significantly altered during 10 mg/kg injection of Ro 47-0203 (71,410 ± 3,285 dyne/cm2) or after ET-1 infusion (77,423 ± 3,297 dyne/cm2).
Pulsatile data
The baseline impedance moduli indicated a first minimum at 2 Hz, which was not changed after administration of 5 mg/kg Ro 47-0203 (Fig 2A). The phase angles also became positive between 1 and 2 Hz. Baseline Zm tended to increase after 5 mg/kg administration of Ro 47-0203. However, the change was not significant. Characteristic impedances between 2 to 7 Hz and 3 to 10 Hz were not significantly changed. There were no significant alterations in the oscillations throughout the higher frequencies in response to 5 mg/kg of Ro 47-0203 injection (Fig 2A).
After 10 mg/kg of Ro 47-0203 neither Zm nor Zo2-7 and Z3-10 Hz were significantly altered. After 10 mg/kg of Ro 47-0203 there was a rightward shift in the first minimum from 1 Hz to 2 Hz (Fig 2B). The phase angles crossed from negative to positive radians at the same frequency of the first minimum. Subsequent infusion of ET-1 did not alter Zm or Zo2-7 and Z3-10 Hz in pigs pretreated with either 5 or 10 mg/kg of Ro 47-0203. The elastic modulus tended to increase in response to ET-1 in 48-hour-old pigs pretreated with 5 mg or 10 mg/kg of bosentan (Table 2). However, the overall change was not significant.
Bosentan (Ro 47-0203)/endothelin-1, 2-week-old pigs
Standard hemodynamics
A 5 mg/kg intravenous bolus of bosentan to 15 pigs produced a significant increase in PAP (p < 0.05) and PVR (p < 0.05; Table 3). However, neither mean PAF nor mean LAP were changed. Subsequent infusion of ET-1 caused a further increase in PAP (Table 3). PVR continued to increase with the subsequent infusion of ET-1. Baseline AP (73,084 ± 2,836 dyne/cm2) was not altered after 5 mg/kg bosentan administration (72,474 ± 3,221 dyne/cm2) or during subsequent infusion of ET-1 (74,937 ± 3,033 dyne/cm2).
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Table 3. Pulmonary Vascular Response to Pretreatment With Bosentan (Ro 47-0203 at 5 mg/kg and 10 mg/kg) Followed by Subsequent Endothelin-1 Infusion in 2-Week-Old Pigs
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After injection of 10 mg/kg of bosentan neither baseline pulmonary artery pressure nor pulmonary vascular resistance were significantly altered (Table 3). PAF and LAP were not significantly altered. Subsequent infusion of ET-1 caused no elevation in PAP or PVR. Both PAF and LAP were unchanged with subsequent infusion of ET-1. Baseline AP (98,107 ± 3,466 dyne/cm2) was not altered after administration of 10 mg/kg of bosentan (93,346 ± 3,124 dyne/cm2) or with subsequent infusion of ET-1 (91,752 ± 2,981 dyne/cm2).
Pulsatile data
The overall shapes of the impedance moduli were not altered after administration of either 5 mg/kg or 10 mg/kg of Ro 47-0203 (Fig 3A and B, respectively). The first minimum and phase angle crossover (negative to positive) occurred between 2 and 3 Hz with either concentration of bosentan. Baseline input mean impedance was significantly elevated (p < 0.05) after 5 mg/kg bosentan administration (Table 3). After administration of 10 mg/kg of bosentan, input mean impedance was not significantly altered (Table 3). Characteristic impedances between 2 to 7 Hz and 3 to 10 Hz were not significantly altered during infusion of either concentration of bosentan.
Subsequent infusion of ET-1 caused a further augmentation in input mean impedance in animals pretreated with 5 mg/kg of bosentan, whereas piglets pretreated with 10 mg/kg of bosentan experienced no change in input mean impedance after ET-1 administration. The elastic modulus in 2-week-old pigs pretreated with either 5 or 10 mg of bosentan was not significantly changed with subsequent infusion of ET-1 (Table 3).
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Comment
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Pressure and resistance response to endothelin-1
ET-1 produced age-dependent responses in the distal pulmonary arterial circulation of the newborn pig. Pulmonary artery pressure was diminished and pulmonary vascular resistance tended to decrease (p = 0.55) in 48-hour-old pigs. In contrast, these parameters were elevated by the same dose of ET-1 in 2-week-old pigs. Our findings are consistent with those of Bradley and colleagues [17] who also demonstrated pulmonary dilation to ET-1 in the newborn pig. Their study was the first to note this unique response in the newborn pig. However, their investigation was limited to pigs less than 10 days old and there was no attempt to occlude the ductus arteriosus to prevent shunting between the systemic and pulmonary circulations. All newborn pigs studied in our investigation had a patent ductus arteriosus that required ligation under direct vision. A patent ductus would directly alter pulmonary hemodynamics. Furthermore, Bradley and colleagues did not consider the elastic modulus or hydraulic impedance alterations that may occur with this potent vasoactive agent. Similarly, Wong and associates [18] found that ET-1 produced a dramatic pulmonary vasoconstrictor response in juvenile sheep (6 to 12 months old), while newborn sheep (< 1 week old) had a minimal response, again supporting that the response to ET-1 is age dependent.
In contrast to the ET-1 induced pulmonary vasodilation reported in the newborn pig lung [4, 5, 17], potent pulmonary vasoconstrictor activity to ET-1 has been observed in a number of adult animal species including the pig [7, 19]. The specific chronologic age during maturation that the pulmonary vascular response to ET-1 changes from dilation to constriction has not been established. Our data indicate that the vascular response to ET-1 changes dramatically within the first 2 weeks of postpartum life and that the pulmonary dilation observed at 48 hours is a transient phenomenon. Newborns quickly lose the ability to dilate in response to ET-1. Instead, pulmonary arterial constriction occurs, reflected by the elevation in pulmonary artery pressure, pulmonary vascular resistance, and input mean impedance in 2-week-old pigs. Unlike the biphasic response to ET-1 (dilation followed by constriction) observed by Lippton and colleagues [8] in the feline pulmonary circulation, the decrease in PAP in response to ET-1 in our 48-hour-old pigs was sustained for the entire 10-minute infusion interval [8].
A higher degree of basal proximal and distal pulmonary arterial tone was observed in the younger 48-hour-old animals. This may in part be responsible for the divergent responses to ET-1. Although it was not the goal of this study to examine intracellular mechanisms that may be responsible for the age-dependent response to ET-1, our data do suggest that these intracellular changes occur in the first 2 weeks of life. There are a number of possible cellular mechanisms that may account for this unique vasodilator response to ET-1. In a separate study on near-term fetal sheep, Wong and colleagues [20] found ETB receptors, rather than ETA receptors, played a more significant role in controlling pulmonary vascular tone in the newborn. Although four main ET receptors have been identified, two of these receptors, ETA and ETB, are thought to be the principal receptors in mammalian tissue [11]. ETA is present in vascular smooth muscle and has a high affinity for ET-1 and ET-2, with a low affinity for ET-3 [10, 12]. ETB receptors are further subdivided according to their function: ETB1 receptors mediate vasodilation and ETB2 receptors mediate vasoconstriction. ETB receptors are present in both the vascular smooth muscle and endothelium and show equal affinity for all three isoforms of endothelin [10, 12]. Our data as well as that of Wong and associates [20] support the hypothesis that a subset of ETB receptors may predominate in the pulmonary circulation during this early newborn interval. It is possible that there is an alteration in receptor density resulting in a decrease in ETB receptors and an increase in ETA receptors with pulmonary maturation.
Response to bosentan (Ro 47-0203)
Bosentan is a novel ET-1 antagonist that acts through competitive inhibition of ETA and ETB receptors [12, 13]. Clozel and colleagues [12, 13] initially reported that bosentan and a similar compound, Ro 46-2005, are both orally and parenterally active. In our study, at both doses, bosentan increased pulmonary artery pressure in 48-hour-old pigs suggesting a slight agonist action. Bosentan, which binds ETA and ETB receptors with nearly equal efficacy, most likely blunted the pulmonary dilator response in 48-hour-old pigs by blocking the ETB receptors.
Similarly, in 2-week-old pigs, 5 mg/kg of bosentan appeared to function as a partial agonist, causing an overall pulmonary arteriolar vasoconstriction reflected by the augmented PAP, PVR, and Zm. However, at 10 mg/kg of bosentan, there were no changes in pulmonary artery pressure, pulmonary vascular resistance, or input mean impedance. Thus, a divergent response was observed at two different doses in 2-week-old pigs. Perhaps at the weaker dose (5 mg/kg), bosentan may have a preferential affinity for one receptor type over the other (eg, ETB receptors), whereas at the more concentrated level both receptor types are more equally bound.
Response to ET-1 subsequent to Ro 47-0203 administration
The pulmonary vasodilator response observed with ET-1 infusion in 48-hour-old pigs was ablated when the animals were pretreated with either concentration of bosentan. In fact, subsequent ET-1 infusion caused pulmonary artery pressure to increase in pigs pretreated with 10 mg/kg of bosentan (Table 2).
Infusion of ET-1 in 2-week-old pigs pretreated with 5 mg/kg of bosentan caused a further increase in PAP and Zm (Table 3). This indicates that the pressor effect of ET-1 was not inhibited with this concentration of bosentan. However, in 2-week-old pigs pretreated with 10 mg/kg of bosentan, ET-1 did not alter mean or pulsatile pulmonary hemodynamics. Thus only at the higher dose was bosentan effective in ablating the ET-1 induced pulmonary arteriolar vasoconstriction. This finding may be useful clinically as pulmonary hypertension in both the infant and adult has been associated with increased levels of endothelin-1.
EY and Zo response to ET-1 and bosentan
Although there is increasing evidence that the vascular response to ET-1 is dependent upon the age of the animal, there have been no reports of the effects of ET-1 on the pulmonary artery elastic modulus or hydraulic impedance response in either newborn or adult animal models. Although there were no significant changes in proximal arterial distensibility or compliance with ET-1, there was a decrease in the oscillations in the impedance moduli throughout the higher frequencies in 48-hour-old pigs. These impedance alterations are the result of the vasodilating activity of ET-1 observed in 48-hour-old pigs. The altered oscillations are indicative of changes in the major wave reflecting sites as well as an altered wave velocity. The impedance moduli in 2-week-old animals were not significantly modified during ET-1 administration. Bosentan by itself had no significant effects on the elastic modulus or characteristic impedance in either the 48-hour-old or 2-week-old pigs. Therefore, the vascular action of both ET-1 and bosentan predominantly affects the distal pulmonary arteriolar bed rather than the larger proximal pulmonary arteries. The lack of proximal pulmonary arterial vascular activity may reflect variations in receptor densities or alternatively the higher degree of preexisting baseline stiffness in the proximal newborn pulmonary vessels. This may make them fairly noncompliant and therefore resistant to alterations in elasticity with exogenous agents.
Calculation of the elastic modulus in the typical fashion (ie, with the upper and lower limits of strain regulated by systole/diastole) defines narrow incremental alterations. In this study the elastic modulus (EYo) was determined with strain normalization to the "resting" or nonstrained radius (Ro). Ro was approximated through linear extrapolation of the high-fidelity pulmonary artery pressure-radius curve to zero pressure during superior and inferior caval occlusion. Although the relationship between pressure and radius is not linear, our calculations demonstrate that the error introduced in treating the lowest point on the curve as linear, at an already significantly diminished pressure, is less than 1% (see Appendix).
Aortic pressure response to ET-1 and Ro 47-0203
Unlike the age-dependent pulmonary arterial responses to ET-1, systemic pressure was elevated by ET-1 in both age groups indicating absence of maturational changes to ET-1 in the systemic circulation. The divergent responses suggest a baseline difference in receptor density or subtype between the two circulations.
At both concentrations, bosentan prevented the ET-1 induced systemic vasoconstriction in younger and older piglets. The systemic data suggest that the vasoconstrictor response to ET-1 works through activation of both ETA and ETB receptors, as inhibition of these receptors with Ro 47-0203 ablated the constrictor response. The varied response between the pulmonary and systemic circulations suggests that other receptors in addition to endothelin may also be involved in the pulmonary arteriolar constrictor response to ET-1.
Conclusions
This study indicates that the effects of endothelin and bosentan are mediated through actions that affect predominantly the distal arteriolar region and not the proximal pulmonary arteries in the lung of both newborn 48-hour-old and 2-week-old Yorkshire pigs. This is supported by the observed alterations in PVR and Zm with no concomitant changes in the elastic modulus or characteristic impedance. However, ET-1 decreased impedance moduli oscillations in the higher frequencies in 48-hour-old pigs suggesting reduced wave reflections. The alterations in the impedance moduli are consistent with pulmonary vasodilation in 48-hour-old pigs and pulmonary arteriolar constriction in 2-week-old pigs in response to ET-1. Pretreatment with bosentan ablated the ET-1 induced pulmonary depressor response in 48-hour-old pigs and completely abolished the ET-1 induced pulmonary arteriolar constriction in 2-week-old pigs. This finding may be useful clinically as ET-1 has been implicated to play a principal role in the pathogenesis of newborn pulmonary hypertension [24]. This study also indicates that the pulmonary vascular response to ET-1 in piglets changes from dilation to constriction within the first 2 weeks of life. [21]
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Acknowledgments
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This study was supported in part by an American Heart (Nation’s Capital Affiliate) grant. The authors of this paper are appreciative of Dr. Martine Clozel for the generous gift of bosentan that made these studies possible.
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Appendix
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Hemodymanics
Pulmonary vascular resistance was calculated in the usual fashion:
where 
PA = mean pulmonary artery pressure; LA = mean left atrial pressure, and 
PA = mean pulmonary artery flow. Pulmonary arterial impedance calculations were based on Fourier analysis of pressure and flow waves as previously described [14, 22]. Data collection periods were 30 seconds and 6 to 10 random heart beats were analyzed for each period. Ten harmonics were calculated for each heart beat.
Total pulmonary flow is expressed:
where Qm = mean flow, Qn = amplitude of the nth harmonic, 
= the fundamental angular frequency (2
f, where f = frequency in Hz), t the length of the sequence, and 
n = phase angle of the nth harmonic.
Pressure waveforms are expressed:
where Pm = mean pressure, Pn = amplitude of the nth harmonic, and ßn = the phase angle of the nth harmonic. Dividing mean pressure by mean flow (Pm/Qm) produces the input impedance (Zm) to mean flow at the zeroth harmonic. Similarly, the division of each of the sinusoidal terms (Pn/Qn) gives the input impedance for the nth harmonic. The corresponding phase angle (
n, depicted in radians in each graph) was calculated from subtraction of the flow phase angle from the pressure phase angle (ßn - n). Characteristic impedance (Zo) is defined as the impedance in the absence of wave reflections and was calculated between 2 to 7 Hz and 3 to 10 Hz. Statistical difference within each group was assessed by a nonparametric two-tailed Mann-Whitney U test.
Elastic modulus from tension/strain curves (EYo)
The elastic modulus defines the relationship between the stress (force/area) placed upon a vessel wall and the resultant deformation in vessel radius (strain = Rx - Ro/Ro). Through simultaneous measurements of pressure and diameter, pressure-radius and tension-strain curves were constructed for all pigs during each data collection interval. Our elastic modulus (EYo) was derived from stress-strain curves. EY is defined as the ratio of stress to strain, where stress = pressure x radius (the radius existing at mean pressure)/h (wall thickness); strain = Rx - R0/R0 where Rx = the measured radius and R0 = the radius existing at zero pressure. R0 was obtained through a linear extrapolation of a pressure-radius curve (obtained during venae caval occlusion) to 0 mm Hg. The elastic modulus calculations assume a pulmonary artery wall thickness of 180 µm for both 48-hour and 2-week-old pigs [23]. This value was obtained from Greenwald and colleagues who determined pulmonary artery wall thickness from isolated vessel rings in a series of newborn pigs. Although it is technically difficult to monitor wall thickness changes in an intact animal model, our calculations show that treating wall thickness as a constant introduces an error of less than 1%.
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During occlusion of both the superior and inferior vena cava, pulmonary artery pressure consistently approached 5000 to 8000 dynes/cm2, leaving are latively short distance for linear extrapolation to Ro. A Dell pentium computer was used to process the data and construct pressure-radius and stress-strain curves.
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Discussion
Ann. Thorac. Surg. 2000 70: 1530.
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Abstract
Introduction
), and during ET-1 infusion (
). Mean moduli ± SEM.
