Introduction

Endothelin (ET) was first isolated, sequenced and cloned by Yanagisawa₁ in 1988. The ET isolated from supernatant of porcine aortic endothelial cells was a peptide of 21 amino acid residues with two disulfide bridges linking CYS3-CYS11. Studies by Inoue and others₂,₃ have revealed the existence of three distinct endothelin (ET) peptides named ET-1, ET-2 and ET-3. ET receptors have been characterized and divided into three subtypes: ET_A (selective for ET-1), ₄ ET_B (equally selective for ET-1, ET-2 and ET-3) ₅ and ETc (selective to ET-3) ₆. Activation of the ET_A receptor is associated with pronounced vasoconstriction, whereas activation of the ET_B receptor occupation is associated with vasodilatation. ETc has been identified although its physiological significance is uncerntain.₇

Endothelin receptors are widely distributed in many tissues and involved in numerous physiologic and pathophysiologic responses.₈ Discoveries of the specific agonists and antagonists for ET receptors are essential for characterization of the responses to endothelins. BQ123 has been proven as the most specific ET_A receptor antagonist₉. IRL1620, a specific ET_B agonist, is vasoactive in some vascular beds.₁₀,₁₁ IRL2500, a small molecular weight compound, is highly selective at attenuating decreases in arterial pressure by ET-1 by blocking the ET_B rceptor.₁₂ Whether or not the pulmonary vasodilator responses to endothelins are mediated by ET_B receptor are unknown. Therefore, the present study was undertaken to determine the influence of IRL2500, a specific ET_B antagonist on the pulmonary vasodilator responses to ET isopeptides. We studied this problem using intact chest rats under conditions of constant pulmonary blood flow.

Materials and Methods

The protocol of the present study was approved by the Animal Research Committee of the Tulane University Medical Center. Male Charles River rats (260-340 gm) were anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg), and allowed to breathe room air enriched with oxygen through a tracheotomy. The anesthetized animals were strapped in a supine position on a fluoroscopic table, and catheters were inserted in the femoral artery for systemic arterial pressure measurement and the femoral vein for drug administration. A specially designed triple lumen balloon catheter was used (Nu-Med, Hopkinton, N.Y.). This catheter is 145 mm in length and 1.1 mm in O.D. It had a specially curved tip to facilitate passage through the right heart and main pulmonary artery, and then into the artery supplying the right lower lobe. At the distal tip of the catheter is a pressure port through which a 0.25 mm soft tip coronary artery angioplasty guide wire is inserted. Two mm proximal to this port is a perfusion port, which permits passage of a 0.34 mm soft-tipped coronary guide wire. A plastic non-dispensable balloon is affixed to a third port just proximal to the perfusion port. When fully distended with contrast material, the balloon is 4.0 mm in diameter and 3.5 mm in length. Before introduction, this catheter curve was initially straightened with 0.45 mm straight wire in the pressure port to facilitate passage from the right jugular vein into the right atrium at the tricuspid valve. As the straight wire was removed, the natural curve permitted easy entry into the right ventricle. The catheter was then passed over a 0.25 mm soft-tipped guiding catheter to the main pulmonary artery and then into the right lower lobe artery.

Mean pressures in the right lower lobe artery and the aorta were continuously recorded. After intravenous injection of Heparin (1000 units/kg), the balloon was then distended with radiopaque material until the lobar arterial pressure fell to pulmonary capillary wedge pressure. The distal portion of the right lower lung lobe was then perfused with blood removed from a carotid artery with an extra corporeal pump (Masterflex Quick-Load Rotary Pump Model #7021-24). The volume of extra corporeal tubing was 1.5 ml. At a perfusion rate of 14.O± 0.62 ml/min, pressure in the perfused lobar artery approximated that in the main pulmonary artery. Since this catheter perfused approximately one-sixth of the lung by weight, this perfusion rate approximated physiologic flow for that lung area. Fig 1.

Figure 1

Fig 1: Experimental Preparation

In order to demonstrate the vasodilator responses the pulmonary vascular bed, the pulmonary vasomotor tone was raised by a continuous intralobar arterial infusion of U46619, a prostaglandin-like substance, at 1.5-2.5 g/min. After pressures were stabilized, intralobar arterial bolus injections of testing agents were given. These agents included endothelin-1 (ET-1), IRL1620 (an ET_B agonist), endothelin-3 (ET-3), as well as several other potent pulmonary vasodilators such as calcitonin-gene-related peptide (CGRP), adrenomedullin (1-52)(ADM-52), a peptide isolated from pheochromocytoma, nitroglycerin (GTN) and isoproterenol (ISO). IRL2500 (ET_B antagonist, 10mg/kg) and BQ123 (ET_A antagonist, 1mg/kg) were given as intravenous injections. ET-1, ET-3, IRL1620, IRL2500, ADM (1-52) and CGRP were purchased from Phoenix Pharmaceutical Inc. BQ123 was bought from RBI, GTN and ISO from Sigma Chemical Company. Data was presented as Mean ± S.E.M. Statistical analysis was done by Student's t-test.

Results

In the intact chest rat model, vasoactive responses to endothelins were studied in the pulmonary and systemic vascular bed under the conditions of constant pulmonary blood flow. Since the pulmonary perfusion flow was kept constant throughout the experiment, the changes in the pulmonary arterial pressure directly reflected the changes in the pulmonary vascular resistance. The mean systemic arterial pressure was 135.0 ± 15.0 mmHg, and did not change significantly throughout the experiments. The baseline pulmonary arterial pressure was 12.8 ± 1.1 mmHg. Following continuous intrapulmonary arterial infusion of U46619, the pulmonary arterial pressure was elevated to 34.8 ± 1.4 mmHg.

Fig 2 illustrates the pulmonary vasodilator responses to ET-1, ET-3, IRL1620 (ET_B agonist), CGRP and rat ADM (1-52) under condition of elevated pulmonary vasomotor tone. All these compounds tested in vivo produced a dose-dependent pulmonary vasodilator response. On the molar bases, CGRP was the most potent endogenous substance, whereas, ratADM (1-52) was the least potent. The pulmonary vasodilator potency order for endothelins was ET-3 > ET-1 > IRL1620. These responses were observed in different animal groups and a limited number of injections were given to each animal to avoid desensitization of ET isopeptides. Injections of ET-1, ET-3 and IRL1620 produced little, if any, pulmonary response at basal vasomotor tone.

Figure 2

Similar to the responses to ET-1, IRL1620, ET-3, the bolus intrapulmonary arterial injections of nitroglycerin (GTN) and isopretenalol (ISO) also produced dose-dependent vasodilator responses in the lung at elevated vasomotor tone. (Fig.3). The pulmonary vasodilator responses to ET-1, IRL1620 and ET-3 were significantly reduced in the animal group pretreated with IRL2500 (ET_B antagonist, 10 mg/kg, i.v.). At a dose of 1ug, ET-1, IRL1620 and ET-3 produced decreases in pulmonary arterial pressure of 8.1 ± 0.4 mmHg, 8.2 ± 0.3 mmHg and 7.9 ± 0.3 mmHg, respectively (n=5, p<0.05 from constricted baseline). In animals pretreated with IRL2500 (ET_B antagonist), the vasodilator responses to ET-1, IRL1620 and ET-3 at the same dose were 0.9 ± 0.1mmHg, 2.1 ± 0.3 mmHg and 1.8 ± 0.3 mmHg respectively, whereas the vasodilator responses to nitroglycerin and isopretanolol (through different mechanisms) were not changed by the pretreatment with IRL2500 (Fig. 3). Injection of IRL2500 alone did not change the control pressure in pulmonary and systemic vascular beds.

Figure 3

Fig 3: At elevated pulmonary vasomotor tone, intra-pulmonary injections of ET-1, ET-3, IRL1620 (ETB agonist), nitroglycerin (GTN) and isopretanolol (ISO) were given in the control group and group of rats pretreated with IRL2500 (ETB antagonist, 10mg/kg, i. v.) for 10 minutes. The vasodilator responses to ET-1, ET-3 and IRL1600, not GTN or ISO were significantly attenuated by the pretreatment of IRL2500.

In another group of rats, pretreatment with BQ123 (an ET_A antagonist, 1mg/kg, i. v.) did not alter the pulmonary vasodilator responses to ET-1, ET-3, IRL1620, nitroglycerin and isopretanolol at elevated vasomotor tone significantly (Fig 4).

Fig 4. At elevated pulmonary vasomotor tone, intra-pulmonary injections of ET-1, ET-3, IRL1620 (ETB agonist), nitroglycerin (GTN) and isopretanolol (ISO) were given in the control group and a group of rats pretreated with BQ123 (ET_A antagonist, 1mg/kg, i. v.) for 10 minutes. BQ123 did not alter the vasodilator responses to these substances in the lung.

Figure 4

Discussion

The present study was the first to investigate the vasodilator responses to endothelins in the pulmonary vascular beds in intact-chest rats under conditions of constant blood flow. The animal model used in the study was developed by the authors and described in previous publications.₁₃,₁₄ A specially designed triple-lumen balloon catheter was used to measure the pulmonary arterial pressure in rats in vivo. The right lower lobe of the lung perfused through the catheter was hemodynamically isolated in the intact chest animal by inflation of the balloon on the tip of the catheter. Since the perfusion flow was kept constant, the change in the pulmonary arterial pressure directly reflected the change in the pulmonary vascular resistance. In order to demonstrate the vasodilator responses to testing compounds in the pulmonary vascular bed, the pulmonary arterial pressure was raised and maintained to certain level by continuous infusion of U46619, a thromboxane analog.

The present data indicates that ET-1, IRL1620, and ET-3 dilate the lung through activation of ET_B receptor since the depressor responses in the pulmonary vascular bed were significantly blocked by IRL2500, an ET_B antagonist. The present study also extends our previous studies₁₅ by demonstrating the existence of ET_B receptor in the pulmonary vascular bed in rats. Endothelins dilate the pulmonary vessels pre-constricted by U46619. However, they do not seem to play much of a role in maintaining the basal vasomotor tone in pulmonary vascular bed, since neither intravenous injection of BQ123 (ET_A antagonist) or IRL2500 (ET_B antagonist) changed the baseline (not pre-constricted) pulmonary pressures. This is consistent with the finding in newborn lamb.¹¹

Distribution of ET receptors varies between species and among tissue types, although it has been generally observed that ET_A receptors predominate in arterial vessels whereas ET_B receptors predominate on the lower pressure side of the circulation. Both receptors are members of the G-protein-coupled family leading to activation of multiple effector pathways. In vascular smooth muscle, an increase in intracellular Ca²⁺ is a common feature occurring after activation of all receptor subtypes.⁷ Gumusel reported that ET-1 and ET-3 promote calcium influx via the L-type calcium channel to promote constriction of pulmonary resistance vessels, whereas activation of potassium channels mediates the vasodilator responses to ET-1, ET-2 and ET-3 in the pulmonary vascular bed in vivo.¹⁵,₁₆

ET receptors are widely distributed in many tissues and involved in numerous physiologic and pathophysiologic responses⁸. Increased plasma concentrations of ET-1 have been described in a variety of diseases, such as pulmonary hypertension, arteriosclerosis, renal failure, acute coronary syndromes, heart failure, migraine and vascular diseases. Recently an increasing number of endothelin receptor antagonists have been synthesized. These antagonists have been shown to inhibit endothelin-mediated vascular responses. Clinical studies are now ongoing to elucidate the pathophysiologic role of endothelins and the potential benefit of the blockade of the system in different disease states.₁₇

Conclusions

In conclusion, the present study was the first to evaluate the pulmonary vasodilator responses to endothelin isopeptides in intact chest rats under conditions of constant pulmonary blood flow. The present data suggests that ET-1, IRL 1620 and ET-3 have similar vasodilator responses in the pulmonary vascular bed by the mechanism through activation of the ET_B receptor. IRL2500 can be a useful pharmacological tool as an ET_B antagonist in the studies of pulmonary vascular regulation in rats in vivo. A better understanding of the hemodynamic effects of endothelins may provide an avenue to new therapeutic options for cardiovascular disorders in the future.

Correspondence to

Qingzhong Hao, MD Department of Anesthesiology, Geisinger Medical Center, 100 N. Academy Avenue, Danville, PA 17822. E-mail: qhao1@geisinger.edu