Ann Thorac Surg 1996;61:1359-1362
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Reactivity to Alpha Agonists Is Heightened in Immature Porcine Pulmonary Arteries
Margit Kadletz, MD,
Rebecca J. Dignan, MD,
Andrew S. Wechsler, MD
Division of Cardiothoracic Surgery, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
Accepted for publication January 5, 1996.
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Abstract
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Background. Pulmonary hypertension after cardiopulmonary bypass is a common problem in pediatric cardiac operations. This study tested the hypothesis that there is a difference between adult and immature pulmonary artery constrictor and dilator responses.
Methods. Reactivity of pulmonary artery ring segments from 22 mature (15 to 19 weeks) and 15 immature pigs (4 to 5 weeks) was tested in a vessel myograph. Potassium as a receptor-independent vasoconstrictor and phenylephrine as an 
-receptor–mediated vasoconstrictor were used to assess smooth-muscle vasoconstriction. To assess endothelial cell function (nitric oxide production and secretion), we used increasing concentrations of bradykinin or acetylcholine. Sodium nitroprusside was used to produce maximum smooth-muscle relaxation at the end of each experiment.
Results. The data demonstrated maturation-independent endothelium and smooth-muscle–mediated vasodilatation. Pulmonary artery ring segments from immature pigs had significantly less KCl constriction compared with mature pigs (p< 0.05). In contrast, pulmonary ring segments from immature pigs demonstrated enhanced -receptor–mediated constriction compared with mature pigs.
Conclusions.These data may explain perioperative pulmonary vasoconstriction in pediatric patients.
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Introduction
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Many arterial components are involved in the regulation of blood pressure. There is a direct response of vascular smooth-muscle cells to different agents and an indirect response mediated by endothelial cells. Most of these reactions are mediated by specific receptors. Smooth-muscle receptor densities within the vascular bed of pulmonary arteries are not homogeneous and exhibit proximal to distal variability [1, 2]. Receptor variability is also found in endothelial cells lining different vascular beds [3]. Maturational differences in receptors also occur [4]. This may explain the variability in adult and perinatal pulmonary vascular hypertension.
The present study tested the hypothesis that intrapulmonary artery ring segments isolated from immature and mature pigs differ significantly in responses to vasoconstrictor agonists. Intrapulmonary arteries of the same size were used in this study to exclude differences resulting from vessel size. Endothelial cell–mediated (nitric oxide–dependent) vasorelaxation as well as pulmonary artery contraction in response to potassium and -receptor agents were compared.
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Material and Methods
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Twenty-two mature (15 to 19 weeks) and 15 immature (4 to 5 weeks) Yorkshire pigs were anesthetized with intramuscular ketamine hydrochloride (25 mg/kg) and intravenous pentobarbital (20 mg/kg). The pigs were mechanically ventilated with room air and a positive end-expiratory pressure of 5 cm H2O. The animals were given heparin (30 U/kg) and the lungs were removed after sternotomy and placed immediately in physiologic saline solution at 4°C.
Studies on Pulmonary Arteries
The first and second branches of the main pulmonary arteries of immature and mature pigs were dissected carefully from the surrounding tissue and cut into 3-mm ring segments.
Rings were suspended on a strain gauge. The upper wire was connected to a Grass FT03D force transducer (Grass Instruments Co, Quincy, MA) and the lower wire was fixed to a micrometer (Mitutyo, Tokyo, Japan). The transducers were calibrated every day. Force responses were recorded on a Grass oscillographic recorder [5]. Ring segments were allowed to equilibrate for 45 minutes in the tissue bath in physiologic saline solution, warmed to 37°C and gassed with 95% O2 and 5% CO2. Indomethacin (10 µmol/L) was added to exclude prostaglandin-mediated reactions. Metoprolol (10 µg/mL) as a ß-receptor antagonist was added to exclude ß-receptor–mediated responses. A normalization procedure was performed as described previously [5]. A passive length-tension curve was obtained for each vessel by separating the wires, and the force generated by the vessel was recorded. The LaPlace formula (P = 2
T/L) was used to calculate pressure from tension. The inner diameter (L = 2pT/30) was measured at a pressure of 30 mm Hg for each vessel. Passive tension was reduced to 90% (L90) before vessel function was tested. Baseline resting pressures (P = 2T/L90) for each group were compared.
Immature and mature pulmonary artery groups were divided into two cohorts. The first cohort of arteries was contracted twice using 125 mmol/L potassium chloride to obtain the maximum possible contraction, followed by a cumulative dose-dependent contraction to phenylephrine. At the end of each experiment, total possible relaxation was obtained in response to 10-4 mmol/L sodium nitroprusside (SNP).
In the second cohort, immature and mature pig pulmonary artery ring segments were contracted twice using 125 mmol/L KCl (total possible contraction), followed by prostaglandin F2 as the contracting agent to 60% to 80% of total possible contraction. Endothelial cell function was tested by using acetylcholine or bradykinin. The drug was added until no further relaxation was obtained. At the end of the experiment, SNP 10-4 mol/L was added to obtain the maximum possible relaxation. Endothelium-mediated response was calculated as a percentage of total possible relaxation to SNP after either bradykinin or acetylcholine.
Mean (± standard error) contraction and relaxation values were calculated for each group and compared between the groups using analysis of variance. Unpaired t test and rank order analysis were used to determine significant differences between groups. Values were considered statistically significant when p was less than 0.05. Only one ring segment from each pig was used for each test series, and each ring segment was used for just one series.
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Results
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There was no difference in the diameter of artery ring segments used for the experiment (2.9 ± 0.1 mm in mature pigs; 2.8 ± 0.2 mm in immature pigs).
Significantly increased maximum contraction to 125 mmol/L KCl (Fig 1
) was obtained in the pulmonary artery ring segments from mature pigs (36.0 ± 1.8 mm Hg) compared with the pulmonary artery rings of immature pigs (28.8 ± 2.0 mm Hg; p > 0.05).
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Fig 1. . Maximal contraction of pulmonary arteries in response to 125 mmol/L potassium, measured in millimeters of mercury. Contraction of mature pulmonary arteries was significantly increased compared with contraction of immature vessels: 36.0 ± 1.8 mm Hg in mature (n = 22) versus 28.8 ± 2.0 mm Hg in immature pigs (n = 15). *p < 0.05.
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Using cumulative concentrations of phenylephrine, there was no difference between mature and immature rings in the percentage of total possible contraction to potassium using low doses of phenylephrine (10-9 to 10-7 mol/L). By increasing the concentration, a significantly greater contraction was obtained in immature compared with mature pulmonary artery ring segments (Fig 2).
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Fig 2. . Cumulative concentrations of phenylephrine, calculated as a percentage of total possible contraction to potassium. There was no difference using low doses (10-9 to 10-7 mol/L). Increasing the concentration of phenylephrine (10-6.6 to 10-4 mol/L) resulted in a significantly different response between immature (n = 13) and mature (n = 20) pig pulmonary artery vessel contractions.
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In contrast to contraction, the total possible smooth-muscle relaxation, obtained at the end of each experiment by adding SNP, was not different between the groups (mature, 108.7% ± 0.3%; immature, 107.7% ± 0.04%) (Fig 3).
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Fig 3. . Maximal relaxation to sodium nitroprusside (SNP) was not different between immature (n = 15) and mature (n = 22) pulmonary artery vessels (107.7% ± 0.3% versus 108.7% ± 0.04%, respectively).
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The other cohort of arteries was used to compare endothelial cell–mediated relaxation, calculated as a percentage of total possible relaxation to SNP. Relaxation to acetylcholine was 91.0% ± 0.03% of total relaxation in pulmonary artery ring segments from mature pigs and 84.1% ± 0.05% of total relaxation in ring segments from immature pigs. The difference was not significant (Fig 4).
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Fig 4. . Relaxation to acetylcholine was calculated as a percentage of total possible relaxation to SNP. In pulmonary artery ring segments from mature pigs (n = 21), it was 91.0% ± 0.03%, and in ring segments from immature pigs (n = 13), it was 84.1% ± 0.05% of total relaxation (p = not significant).
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Comparing endothelial cell–mediated relaxation in response to bradykinin, there was also no difference obtained between pulmonary artery ring segments from mature pigs (93.3% ± 0.04%) and immature pigs (98.5% ± 0.01%) (Fig 5).
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Fig 5. . Endothelial cell–mediated relaxation to bradykinin, calculated as a percentage of total relaxation to KCl, was not different between pulmonary artery ring segments from mature pigs (n = 22; 93.3% ± 0.04%) and immature pigs (n = 14; 98.5% ± 0.01%).
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Comment
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Pulmonary hypertensive crises are relatively common in high-risk infants after operations for congenital heart disease [6]. Hypoxic stimulation of the precapillary arteriolar resistance region is perhaps the most potent vasoconstrictor. This can be caused by inadequate alveolar oxygen concentrations, reduced mixed venous oxygen saturation, or arterial desaturation. Hypoxic contraction was excluded in our experiments by using an ex vivo model and by keeping the organ bath solution at constant oxygen tension at 37°C. Physiologically, the neonatal pulmonary circulation is more sensitive to catecholamines, both exogenous and endogenous [7, 8].
The results of this study demonstrate an increased response of arterial pulmonary vessels of immature pigs to phenylephrine as compared with mature pulmonary artery vessels. In contrast to these findings, contraction to potassium was significantly increased in mature compared with immature artery ring segments.
The contractile response to potassium is a receptor-independent response of vascular smooth-muscle cells. Contraction is proportional to drug concentration, with an upper limit, and is dependent on the number of smooth-muscle cells. An increased number of smooth-muscle cells in pulmonary artery ring segments of mature pigs seems to be an explanation for the increased contraction to potassium, assuming that the myocytes are the same size or an increased size and of the same number. Therefore, the increased response to phenylephrine found in immature pulmonary artery ring segments cannot be explained by an increased number or size of smooth-muscle cells.
The increase in vascular sensitivity in the immature pulmonary artery ring segments may reflect an increase in the effective number of active -receptor sites per muscle mass or an increase in agent–receptor binding efficiency. Enhancement in the ability of smooth muscle to generate tension is unlikely, because the receptor-independent response to potassium was significantly increased in mature pulmonary artery ring segments.
Because pulmonary arterial adrenergic sensitivity is predominantly -mediated, catecholamines with mixed and ß activity tend to have constrictor effects in the pulmonary circulation [7–9]. In preliminary studies, the vascular response to ß agents was tested, but constant relaxation could not be obtained in immature or mature pig pulmonary artery ring segments. To exclude a ß-receptor–mediated response, we added a high dose of ß-antagonist to the organ bath solution during calibration for 45 minutes.
Smooth-muscle relaxation to SNP is also receptor independent. Nevertheless, there was no difference in total possible relaxation between immature and mature pulmonary artery ring segments. Sodium nitroprusside was used at the end of each experiment, in addition to nitric oxide–stimulating agents, to obtain the maximum possible relaxation. Nitric oxide is also the active agent of SNP. Therefore, a difference in smooth-muscle–cell responses to nitric oxide can be excluded. We also studied the response to acetylcholine, mediated by muscarine receptors, and to bradykinin, mediated by bradykinin receptors. Receptors found on the endothelial cell surface seem to be fully developed in pulmonary arteries from immature pigs compared with mature pigs. There was no difference found either in acetylcholine- or in bradykinin-stimulated, nitric oxide–mediated relaxation.
These data demonstrate maturation-independent, endothelial and smooth-muscle–mediated vasodilatation. Immature segments had diminished direct KCl constriction but strongly enhanced -receptor–mediated constriction. Pulmonary artery function was tested in vitro, excluding intraoperative factors such as high flow and pressure. Nevertheless, this observation may help explain perioperative pulmonary vasoconstriction in pediatric patients.
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Acknowledgments
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This investigation was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung J0572-MED, Austria; and by the National Heart, Lung, and Blood Institute, 5 R01 HL26302.
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Footnotes
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Address reprint requests to Dr Wechsler, Department of Surgery, Medical College of Virginia, MCV Station Box 645, Richmond, VA 23298-0645.
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References
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Abstract
Introduction
