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NEUROSURGICAL ANESTHESIA

The Hemodynamic Effects of Endothelin Receptor Antagonism During a Venous Air Infusion in Dogs

Jose Eduardo Tanus-Santos, MD, PhD^*, Wladimir Mignone Gordo, PharmD, MSc^*, Artur Udelsmann, MD, and Heitor Moreno Junior, MD, PhD^*

Departments of *Pharmacology and Anesthesiology and Research Laboratory of Anesthesia, State University of Campinas, São Paulo, Brazil

Address correspondence and reprint requests to Jose Eduardo Tanus-Santos, MD, PhD, Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, PO Box 6111, 13083-970 Campinas, São Paulo, Brazil. Address e-mail to tanus@ unicamp.br.

	Abstract

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Endothelin (ET) is involved in the humoral component of the vasoconstriction during pulmonary embolism. We examined the effects of selective ET receptor antagonists on the hemodynamic and respiratory changes and on serum thromboxane B₂ (TXB₂) levels, during a continuous venous air infusion (VAI) in anesthetized mongrel dogs. The VAI (0.2 mL · kg^-1 · min^-1) was initiated 5 min after an injection of saline (controls, n = 7), 1 µmol of the selective ET_A receptor antagonist JKC-301 (group A, n = 6), or 1 µmol of the selective ET_B receptor antagonist BQ-788 (group B, n = 6). Hemodynamic evaluation was performed every 15 min of VAI, and blood samples were drawn for blood gas analysis and TXB₂ determinations. The increase in pulmonary perfusion pressure after 30 min of VAI was attenuated in Group A compared with the controls and Group B (Group A = 7 ± 1 mm Hg; Group B = 16 ± 1 mm Hg; controls = 14 ± 1 mm Hg; P < 0.05). Pulmonary vascular resistance showed a similar behavior. TXB₂ concentrations increased after 60 min of VAI in the controls and in Group B, but not in Group A (controls = 48%; Group B = 104%; Group A = 18%; P < 0.05 for controls and Group B). Similar decreases in PaO₂ and SaO₂ were observed in the three groups. We conclude that antagonism of ET_A receptors attenuates the hemodynamic changes and blunts the increase in thromboxane A₂ production during a VAI in dogs.

Implications: We evaluated the effects of endothelin receptor antagonists during a venous air infusion in dogs. Endothelin_A receptor antagonism attenuated the hemodynamic changes and blunted the increase in thromboxane A₂ production in this setting.

	Introduction

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The increase in pulmonary vascular resistance during a pulmonary embolism (PE) is caused mainly by pulmonary arterial obstruction to blood flow and by the release of vasoactive substances (1). Endothelin 1 (ET 1), a member of the endothelin family (2), is involved in the humorally mediated component of the vasoconstriction and cardiodepression during PE (3). Abnormalities in ET 1 metabolism ensue (4), and the resulting higher circulating levels of this peptide reflect the pulmonary endothelial cell dysfunction which occurs in acute PE and in pulmonary hypertension (5).

ET 1 may also increase pulmonary vascular resistance after experimental pulmonary air embolism (6,7). However, these studies were performed in rabbit isolated perfused lungs, and conflicting results were found regarding this issue (6,7). Furthermore, because of methodological limitations, these studies did not evaluate whether ET 1 affects the hemodynamic responses of the cardiovascular system as a whole during pulmonary air embolism. In addition, the use of perfusion solutions did not consider that platelets and granulocytes can also release vasoactive substances such as serotonin, thromboxane A₂ (TXA₂), and ET 1 during acute lung injury (8,9).

Because the role of ET 1 in the cardiovascular responses to pulmonary air embolism is not clear and ET_B receptors are exclusively responsible for pulmonary ET 1 removal in dogs (10), we examined the effects of selective ET_A and ET_B receptor antagonists on the hemodynamic and respiratory changes induced by a continuous venous air infusion (VAI) in dogs. Because it is possible that ET 1 released after VAI stimulates the formation of TXA₂ by pulmonary tissue (11), we also measured the serum concentrations of TXB₂ (the stable breakdown product of TXA₂) to assess the effects of selective ET receptor antagonism on the in vivo production of TXA₂, a known pulmonary vasoconstrictor (12), during a VAI.

	Methods

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All procedures were approved by our animal care committee. The animals were handled according to the guideline principles published by the National Institutes of Health and the Council of the American Physiology Society. Nineteen mongrel dogs (10.4 ± 0.6 kg) of either sex were anesthetized with pentobarbital (30 mg/kg, IV) and pancuronium (0.1 mg/kg, IV) and tracheally intubated, and their lungs were mechanically ventilated with room air using a volume-cycled respirator. The tidal volume was 15 mL/kg, and the respiratory rate was adjusted to maintain a baseline (BL) physiological PaCO₂. Anesthesia was maintained with an infusion of pentobarbital (6–10 mg · kg^-1 · h^-1). Fluid-filled catheters were placed into the right femoral artery and right femoral vein for mean arterial pressure (MAP) monitoring via a pressure transducer and fluid administration, respectively. A 7F, flow-directed pulmonary catheter was placed into the pulmonary artery via the right external jugular vein, and its correct location was confirmed by detection of the typical pressure wave of this artery. The catheter was connected to a pressure transducer to allow the monitoring of mean pulmonary artery pressure (MPAP), central venous pressure, and pulmonary capillary wedge pressure (Pcwp). The transducers were zeroed at the level of the right heart and recalibrated before each set of measurements. Cardiac output was determined in triplicate by injecting 5 mL of saline, and the results were recorded on a computerized system. The heart rate (HR) was measured using a surface electrocardiogram (lead I).

After at least 20 min of stabilization, a BL hemodynamic evaluation was performed, and the animals were randomly assigned to one of three experimental groups: dogs in Group A (n = 6) received a bolus injection of 1 µmol of the selective ET_A receptor antagonist JKC-301 (Sigma); dogs in Group B (n = 6) received a bolus injection of an equimolar dose of the selective ET_B receptor antagonist BQ-788 (Sigma, St. Louis, MO). This dose of BQ-788 (1 µmol) had no significant hemodynamic effect and completely abolished ET 1 removal by the lung in dogs (10). Dogs in the control group (n = 7) received the same volume of saline. Hemodynamic data were recorded 5 min after the administration of the antagonists (or saline). Thereafter, a continuous VAI, 0.2 mL · kg^-1 · min^-1, was initiated in all animals via the femoral vein and was maintained throughout the experiment. Hemodynamic data were recorded every 15 min (15, 30, 45, and 60 min time points) after beginning the VAI. The cardiac index (CI), systemic vascular resistance index (SVRI), and pulmonary vascular resistance index (PVRI) were calculated by using standard formulas. Blood samples were drawn from the femoral artery at BL, 30, and 60 min for blood gas analysis. PaO₂, SaO₂, PaCO₂, arterial blood pH, and hemoglobin were determined. Plasma TXA₂ levels (measured as TXB₂) were determined in arterial blood samples (drawn at BL and 60 min) using a commercial enzyme immunoassay (Cayman Chemical Co., Ann Arbor, MI).

The results are expressed as mean ± SEM. The changes in PVRI, pulmonary perfusion pressure (MPAP-Pcwp), CI, MAP, SVRI, and HR are presented as the difference between the BL value and the value recorded at each time point. Changes from BL values and comparisons among groups at each time point were analyzed using one-way analysis of variance for repeated measures followed by the Student-Newman-Keuls test. A probability value < 0.05 was considered the minimal level for statistical significance. The sample size of the current study was sufficient to detect differences in pulmonary perfusion pressure and PVRI at a significance level of 0.05 with the power of 70%–80%.

	Results

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There were no significant differences in the BL hemodynamic and respiratory variables among the three experimental groups (Tables 1 and 2). The hemodynamic variables remained unchanged after the administration of saline or ET receptor antagonists (Figs. 1). and 2

View larger version (22K): [in this window] [in a new window]   Figure 1. Change in pulmonary vascular resistance index (PVRI), pulmonary perfusion pressure, and cardiac index (CI) in Group A (), Group B (•) and controls () 5 min after the injection of ET antagonists or saline (ANTAG time point) and after 15, 30, 45, and 60 min of VAI. BL = baseline. Values are mean ± SEM. *P < 0.05 versus corresponding BL values for controls, Group A, and Group B. #P < 0.05 Group A versus Group B. +P < 0.05 Group A versus Group B and Group A versus controls.

View larger version (20K): [in this window] [in a new window]   Figure 2. Change in mean arterial pressure (MAP), systemic vascular resistance index (SVRI), and heart rate (HR) in Group A (), Group B (•), and controls () 5 min after the injection of ET antagonists or saline (ANTAG time point) and after 15, 30, 45, and 60 min of VAI. BL = baseline. Values are mean ± SEM. *P < 0.,05 versus corresponding BL values for controls and Group B.

No hemodilution resulted from repetitive saline injections, as revealed by the sustained hemoglobin levels throughout the study period (Table 2). The VAI induced similar decreases in PaO₂ and SaO₂ in the three groups (Table 2). However, the mild VAI-induced decrease in pH and increase in PaCO₂ were attenuated in animals pretreated with the ET_A receptor antagonist when compared with the other two groups (Table 2).

TXB₂ concentrations increased after 60 min of VAI in the control group and in animals pretreated with the ET_B receptor antagonist, but not in those pre-treated with the ET_A receptor antagonist (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Figure 3. Serum thromboxane B2 concentrations at baseline (BL) and after 60 min of VAI. Values are mean ± SEM. *P < 0.05 versus corresponding BL values. #P < 0.05 Group A versus Group B.

	Discussion

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ET 1 is a potent pulmonary vasoconstrictor released during pulmonary air embolism (13). This peptide activates ET_A and ET_B receptors on pulmonary vascular smooth muscle cells causing contractile responses (14,15) and may stimulate the release of TXA₂ from pulmonary tissue (16). In agreement with these observations, high levels of ET 1 and TXB₂ were observed after an experimental VAI (13,17). Because these humoral factors contribute to the mechanical obstruction in causing pulmonary hypertension (14), the use of pulmonary vasodilators (18) or the pharmacological antagonism of these mediators may have beneficial hemodynamic effects in PE.

Our results suggest that the antagonism of ET_A receptors attenuates the hemodynamic changes in a VAI by reducing the pulmonary vasoconstrictor effects of ET 1 acting on ET_A receptors (19). In addition, it was previously suggested that ET 1 activates the cyclooxygenase pathway resulting in increased TXA₂ levels (6). Thus, it is possible that ET_A receptors antagonism attenuated the pulmonary vasoconstriction after the VAI by blunting the increase in TXA₂ production. Previous studies using another specific ET_A antagonist (LU135252) showed either no effect (7) or a reduced pulmonary vascular reaction after air embolism (6). These conflicting studies were performed in rabbit isolated perfused lungs and may not reflect the pathophysiological features found in intact animals. For example, thrombocytopenia may occur as a result of activated platelet sequestration at the air-blood interface during gas embolism (20). Because perfusion studies use buffer solutions to perfuse the lungs, platelet activation and increased TXA₂ release (21) are not taken into consideration. The increased TXA₂ concentrations in perfusion solutions after air embolism (6) may thus underestimate the real increase in TXA₂ concentrations in intact animals. Although we did not measure the levels of ET 1, the lower PVRI and pulmonary perfusion pressures found in dogs treated with the ET_A receptor antagonist confirmed an important role for ET_A receptors in mediating the pulmonary vasoconstriction and the increase in total TXA₂ production after a VAI.

Endothelial ET_B receptors are responsible for pulmonary ET 1 removal in dogs (10). The dose of the specific ET_B receptor antagonist (BQ-788) we used (1 µmol) completely abolished the removal of ET 1 during a single pulmonary transit time in dogs (10). Thus, the blockade of ET_B receptors may increase the amount of ET 1 available to act on ET_A receptors and explains the similarity in the hemodynamic responses and changes in TXB₂ concentrations between the controls and ET_B receptor antagonist-pretreated dogs.

Hypoxemia and CO₂ retention were detected in the three experimental groups (Table 2). Small amounts of continuous gas infusion (0.2 mL · kg^-1 · min^-1) increase high ventilation/perfusion areas in the lung (22). However, the VAI-induced hypoxemia results from an increased venous admixture arising from lung units with low ventilation/perfusion ratios (20), and this effect of the VAI was apparently not influenced by ET receptors antagonists. The analysis of alveolar-arterial O₂ tension difference indicates that average tension difference values for controls at 30 and 60 min (37 and 31 mm Hg) were lower than those of group A (49 and 44 mm Hg) and group B (39 and 42 mm Hg). This finding suggests that the oxygenating capacity of the lungs was adversely affected by both ET receptor antagonists.

The decrease in SVRI and MAP after the VAI in the controls and ET_B receptor antagonist-pretreated dogs may be only partially explained by the higher PaCO₂ and lower pH levels in these two groups compared with animals pretreated with the ET_A receptor antagonist. In this regard, ET receptors antagonists may have influenced the mechanisms whereby ET 1 acts at the central nervous system (23) and affects sympathetic nervous activity and other neurohumoral mechanisms for cardiovascular homeostasis.

The lower increases in PVRI and pulmonary perfusion pressure observed after VAI in group A gives support to the hypothesis that ET 1 is an important mediator released during air embolism (13). However, other humoral mediators (TXA₂, serotonin, histamine), the mechanical obstruction of pulmonary vessels, and general arteriolar neurogenic vasoconstriction contribute to the pulmonary hypertension PE (1,14,20).

In conclusion, the antagonism of ET_A (but not ET_B) receptors attenuates the hemodynamic changes and blunts the increase in TXA₂ production during a VAI in dogs.

	Acknowledgments

 
This work was supported by Fundação de Amparo a Pesquise do Estado de São Paulo, Brazil.

The authors thank the Center of Experimental Medicine and Surgery (UNICAMP) and Dr. Stephen Hyslop for reviewing the manuscript.

	Footnotes

 
JET-S and WMG are supported by FAPESP-SP.

	References

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