irl-1620 and Hypoxia

Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ETB) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (approximately 0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ETB receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg Po2) compared with normoxic control (140 mmHg Po2). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H2O2. The hypoxic effect on ETB receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-L-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H2O2, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ETB receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ETB receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ETB receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ETB receptors on smooth muscle.

Topics: Animals; Down-Regulation; Endothelin-1; Endothelins; Endothelium, Vascular; Hypertension, Pulmonary; Hypoxia; Lung; Male; Muscle Contraction; Muscle, Smooth, Vascular; Peptide Fragments; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptor, Endothelin B; Superoxides; Vasoconstriction

Endothelin-1 (ET-1) is a potent vasoactive peptide, and is thought to play an important role in the regulation of pulmonary vascular tone. Previous studies suggested that ET-1 can act as a vasoconstrictor via the endothelin-A and -B 2 receptors located on smooth muscle cells, and also act as a vasodilator through the endothelin-B 1 receptor situated on endothelial cells in the pulmonary circulation. To determine the role of endothelin-B receptors in hypoxic pulmonary hypertension, we examined the hemodynamic effects of a selective endothelin-B receptor agonist (IRL 1620) in chronic hypoxic pulmonary hypertensive rats. In rat lungs perfused with a half-blood solution, vasoconstriction by KCl administration was gradually reversed by IRL 1620 in normoxic rats, but not in chronic hypoxic rats. In in vivo studies, small doses of IRL 1620 induced transient vasodilation in normoxic rats, but had no obvious effects in chronic hypoxic rats. A high dose of IRL 1620 mediated vasoconstriction only in chronic hypoxic rats. Endothelin-B receptor messenger-RNA expression was lower in the lungs of chronic hypoxic rats than in normoxic rats. These results suggested that although the vasodilatory response mediated by endothelin-B receptor can be expected to play a protective role in the development of pulmonary hypertension, that response is diminished in hypoxic pulmonary hypertension.

Topics: Animals; Endothelins; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Male; Peptide Fragments; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Receptors, Endothelin; Vasodilation

Endothelins are potent vasoactive peptides that bind to their specific receptors, playing an important role in the CNS under physiological and pathophysiological conditions. Astrocytes, which have been shown to express these receptors, also have a considerable role to play under physiological and pathophysiological conditions, particularly those involved in delayed neuronal death. We carried out in vitro receptor autoradiographic binding experiments using specific ligands for endothelin receptors on cultured rat astrocytes. On astrocytes, the specific binding sites for (125)I-PD151242 (a selective endothelin A receptor antagonist) and (125)I-IRL 1620 (a selective endothelin B receptor agonist) were detected. We also characterized the qualitative and quantitative changes of endothelin receptors 24 h after subjecting cultured rat astrocytes to a transient 4-h hypoxia/hypoglycemia insult, used as a model of delayed neuronal death. After transient hypoxia/hypoglycemia, the number of endothelin B receptors increased significantly on cultured astrocytes, but this did not occur among the endothelin A receptors. These findings suggest that the astrocytic effects associated with endothelin in delayed neuronal death include gliosis or the repair process or both, manifested primarily by an increase in the number of endothelin B receptors. This rise does not require interaction with other types of CNS cells. Endothelin A receptors might have a role taking their number into consideration on rat astrocytes under both physiological and pathophysiological conditions.

Topics: Animals; Astrocytes; Autoradiography; Azepines; Binding Sites; Cell Death; Cells, Cultured; Endothelin Receptor Antagonists; Endothelins; Hypoglycemia; Hypoxia; Oligopeptides; Peptide Fragments; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Time Factors; Up-Regulation

To investigate whether chronic hypoxia affects endothelin-B (ETB) receptor-mediated pulmonary vasodilation, we compared the vasodilator responses to IRL-1620, a selective ETB-receptor agonist, in isolated perfused lungs from normoxic and chronically hypoxic adult male rats. IRL-1620 caused a dose-dependent vasodilation that was greater in the hypertensive lungs than in the normotensive lungs. In normotensive lungs, a nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine (L-NNA; 300 microM), and an ATP-sensitive potassium (KATP)-channel inhibitor, glibenclamide (Glib; 10 microM), each reduced the vasodilator response to IRL-1620 (1 nM), but the combination of L-NNA and Glib inhibited it more effectively than either drug alone. In contrast, L-NNA alone, but not Glib alone, completely blocked IRL-1620-induced vasodilation in hypertensive lungs. The vasodilator response to a KATP-channel opener, NIP-121 (1 microM), but not the response to sodium nitroprusside (1 microM), was enhanced in hypertensive lungs. We also found increased expression of mRNA for the ETB receptor in lung tissue after hypoxic exposure. In addition, semiquantitative immunohistochemistry demonstrated higher expression levels of ETB receptors in the endothelium of distal segments of the pulmonary artery in hypoxic than in normoxic rats. These results suggest that ETB receptor-mediated pulmonary vasodilation is augmented after chronic hypoxic exposure and that release of NO may be the sole mechanism of this vasodilation in hypertensive lungs, whereas both release of NO and activation of KATP channels are involved in normotensive lungs. We speculate that the underlying mechanism responsible for this augmentation may partly be related to upregulation of ETB receptors in the endothelium of pulmonary resistance arteries in hypertensive lungs.

Topics: Adenosine Triphosphate; Animals; Chronic Disease; Endothelins; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Lung; Male; Nitric Oxide; Nitric Oxide Donors; Peptide Fragments; Perfusion; Potassium Channel Blockers; Potassium Channels; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Vasodilation; Vasodilator Agents

1. Using isolated pulmonary resistance vessels from mature fetal lamb and chronically instrumented lambs (8-17 days old), we have examined whether hypoxic pulmonary vasoconstriction is sustained by activation of a constrictor mechanism or suppression of a dilator mechanism. 2. Hypoxia contracted both arteries and veins in vitro, and the contraction was greater with the former. After removing the endothelium, arteries responded faster to hypoxia, but the magnitude of the response remained unchanged. 3. Hypoxic arteries, unlike normally oxygenated arteries, did not contract with either indomethacin (2.8 microM) or N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). The same vessels relaxed with sodium nitroprusside (SNP, 0.001-10 microM) but not with bradykinin (0.1-100 nM). 4. Endothelin-1 (ET-1, 0.01-10 nM) contracted isolated arteries and veins under normoxic and hypoxic conditions. In both vessels, the contraction was fast in onset and subsidence, and was inhibited by the ETA receptor antagonist BQ123 (1 microM). The ET-1 precursor, big ET-1 (100 nM), also contracted arteries and veins, but compared with ET-1 its action was slower in development. Big ET-1 contraction, unlike ET-1 contraction, was curtailed by the inhibitor of the ET-1-converting enzyme, phosphoramidon (50 microM). 5. ET-1 (0.1-10 nM) had no effect on isolated arteries precontracted with a thromboxane A2 (TXA2) analogue (ONO-11113) and treated with BQ123 (10 microM). Under the same conditions, ET-1 relaxed the veins. Accordingly, in the absence of BQ123 treatment, the selective ETB receptor agonist IRL-1620 (0.1-100 nM) relaxed the contracted veins but not the arteries. 6. BQ123 (10 microM) inhibited the constriction of isolated arteries and veins to hypoxia. Likewise, in the conscious lamb a bolus of BQ123 (0.4 mg kg-1, injected into the pulmonary artery) curtailed the rise in pulmonary vascular resistance (Rpa) brought about by alveolar hypoxia without changing significantly systemic vascular resistance (Rao). Under normoxia, Rpa was insignificantly affected by BQ123. 7. The results indicate that pulmonary resistance arteries are more susceptible to hypoxia than the veins, and that hypoxic vasoconstriction does not require an intact endothelium to occur. Hypoxic tone is ascribed primarily to intramural generation of ET-1, while removal of the tonic action of a relaxant may only have an accessory role in the response.

Topics: Animals; Arginine; Bradykinin; Endothelins; Endothelium; Female; Hypoxia; Indomethacin; NG-Nitroarginine Methyl Ester; Nitroprusside; Peptide Fragments; Peptides, Cyclic; Pregnancy; Pulmonary Circulation; Sheep; Thromboxane A2; Vasoconstriction