Pharmacology of two novel mixed ETA/ETB receptor antagonists, BQ-928 and 238, in the carotid and pulmonary arteries and the perfused kidney of the rabbit.

1. In the present study, we have pharmacologically characterized two novel mixed endothelin ETA/ETB receptor antagonists, namely BQ-928 and BQ-238, in ETA and ETB preparations, the rabbit carotid artery (RbCA) and the rabbit pulmonary artery (RbPA), respectively. These two antagonists were compared to established ETA (BQ-123 and BMS 182874), ETB (BQ-788) and mixed ETA/ETB (SB 209670) receptor antagonists. 2. In the RbCA, the ETA monoreceptor preparation, BQ-238 and BQ-928 had apparent affinities (pA2) of 7.42 +/- 0.22 and 7.22 +/- 0.18, respectively, BQ-788 being inactive in this preparation. In the ETB monoreceptor preparation, the RbPA (when IRL-1620 was used as an ETB receptor agonist), the pA2 for BQ-238 was 7.05 +/- 0.14 and for BQ-928 was 8.43 +/- 0.04. BQ-123 and BMS 182874 were inactive in this preparation. Similar to SB 209670, BQ-238 but not BQ-928 had a higher affinity for the ETA than the ETB receptor. 3. All of the antagonists were tested for their ability to block and reverse endothelin-l-induced vasoconstrictions in the rabbit perfused kidney. In this preparation endothelin-1-induced increases in vascular resistance have been shown to be mediated solely by ETA receptors. All compounds (except BQ-788) blocked the pressor effects of endothelin within the kidney; the calculated IC50 values for BQ-123, BMS 182874, SB 209670, BQ-928 and BQ-238 were 0.4 microM, 2 microM, 0.01 microM, 0.4 microM and 0.09 microM, respectively. 4. In all experiments in the rabbit perfused kidney, endothelin-1 was readministered for a third time, 60 min following cessation of infusion of the above-mentioned antagonists. The response to the third infusion of endothelin-1 following cessation of infusion of BQ-123, BMS 182874 and SB 209670 was not significantly different from that to the third infusion of endothelin in control conditions. However, the response to endothelin-1 was significantly higher than control in tissues pre-infused with BQ-788 or BQ-928 (56 +/- 9 and 41.6 +/- 15%, respectively, n = 8 each, P < 0.05). 5. Our results suggest that in a system where ETA receptor activation is responsible for vasoconstriction and ETB-receptor activation for vasodilatation. ETA receptor selective antagonists or mixed ETA/ETB receptor antagonists which possess high affinity for ETA receptors do not induce hyperresponsiveness to endothelin-1. In contrast, ETB selective antagonists or mixed antagonists possessing a high affinity for ETB receptors (such as BQ-928) interfere with the ETB-receptor-dependent physiological antagonism of endothelin-1-induced pressor responses in these same tissues.

Block of endothelin-1-induced release of thromboxane A2 from the guinea pig lung and nitric oxide from the rabbit kidney by a selective ETB receptor antagonist, BQ-788.

1. The present study characterizes the receptors responsible for endothelin-1-induced release of thromboxane A2 from the guinea pig lung and of endothelium-derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ-123, and a novel selective ETB receptor antagonist, BQ-788. 2. In the guinea pig perfused lung, endothelin-1 (ET-1) (5 nM) induced a marked increase of thromboxane A2 which was reduced by 17 +/- 5.0, 70 +/- 1.0 and 93 +/- 1.2% by BQ-788 infused at concentrations of 1, 5 and 10 nM respectively. In contrast, BQ-123 (0.1 and 1.0 microM) had little or no effect on the ET-1-induced release of thromboxane A2. 3. In the same perfused model, the selective ETB agonist, IRL 1620 (50 nM), stimulated the release of thromboxane A2, but not prostacyclin. The eicosanoid-releasing properties of IRL 1620 were abolished by BQ-788 at 10 nM, yet were unaffected by BQ-123 (1 microM). 4. In the rabbit perfused kidney, BQ-788 (10 nM) potentiated the increase of perfusion pressure induced by endothelin-1 (1, 5 and 10 nM) by approximately 90%, but not that induced by angiotensin II (1 microM). Furthermore, the selective ETB receptor antagonist did not reduce the release of prostacyclin triggered by either peptide. 5. In another series of experiments, pretreatment of the perfused kidney with a nitric oxide synthase inhibitor, L-NAME (100 microM), potentiated the pressor responses to both endothelin-1 and angiotensin II. Under L-NAME treatment, BQ-788 did not further potentiate the pressor response to endothelin-1. 6 Our results illustrate the predominant role of ETB receptor activation in the release of thromboxane A2 and nitric oxide triggered by endothelin-l in the guinea pig perfused lung and rabbit kidney respectively.

Pharmacological properties of endothelins and big endothelins in ketamine/xylazine or urethane anesthetized rats.

Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor peptide formed through a specific conversion of its intermediate precursor, big ET-1, by an endothelin-converting enzyme (ECE). The present study evaluates the capacity of the ECE to convert the three big endothelins (big ET-1, big ET-2, and big ET-3), by comparing the pressor responses to these peptides with those induced by their respective metabolites (ET-1, -2, and -3) in the rat in vivo, anesthetized either with a mixture of ketamine/xylazine or with urethane. The mean basal arterial pressure under urethane anesthesia was not significantly different from that of ketamine/xylazine-treated animals (90/15 mg/kg; intramuscularly), although the basal heart rate was significantly higher in the former animals (urethane: 407 +/- 10 beats/min, ketamine/xylazine: 276 +/- 4 beats/min, P < .01; n = 8 to 17). In ketamine/xylazine and hexamethonium-treated rats (5-min infusion, 10 mg/kg intravenously), intravenous injection of ET-1 (1 nmol/kg) and big ET-1 (1 nmol/kg) induced potent vasopressor effects which lasted for more than 20 min. ET-2 (1 nmol/kg) produced similar pressor responses while big ET-2 (1-37) and big ET-2 (1-38) were twofold less potent than ET-2 (P < .05; n = 3 to 4). Big ET-3 induced a pressor effect only at 4 nmol/kg and was found to be at least 10 times less potent than ET-3. In animals anesthetized with urethane (1.5 g/kg intraperitoneally), the pressor responses induced by the endothelins and their intermediate precursors, as well as the pressor responses to angiotensin II and norepinephrine, were reduced by more than 60% (P < .01) when compared to ketamine/xylazine-treated animals. Big ET-3 was found inactive under urethane anesthesia. Ganglion blockade by hexamethonium did not affect the response to ET-1, big ET-1, ET-3, or big ET-3 in rats anesthetized with either ketamine/xylazine or urethane. On the other hand, big ET-2 (1-38), in contrast to ET-2 or big ET-1, did not release prostacyclin from the rat perfused lung, thus indicating that big ET-2 (1-38) is poorly converted in the pulmonary vasculature, and that the phosphoramidon-sensitive ECE responsible for the pressor effects of big ET-2 is localized elsewhere in the systemic circulation. Our results also show that the choice of anesthetics is crucial for the proper monitoring of the pressor responses to endothelins as well as other pressor agents. Nonetheless, even in what we consider as optimal conditions of anesthesia (threshold dose for the pressor response to ET-1 in ketamine/xylazine-treated rats: 0.01 nmol/kg), big ET-3 remains far less active than big ET-1 as a pressor peptide in the rat, suggesting a preferential processing of the latter by the ECE.

Hyperresponsiveness to ET-1 after treatment with a mixture of ETA and ETB receptor antagonists in the rabbit in vivo and in vitro.

We tested the reversibility of the response to endothelin-1 (ET-1) in the rabbit renal vasculature after termination of an infusion of either a selective ETA receptor antagonist, BQ-123, a selective ETB receptor antagonist, BQ-788, or a mixture of both antagonists. Previous studies from our laboratory have shown that, as in humans, vasoconstriction of the rabbit renal vasculature induced by ET-1 is mediated solely via the activation of ETA receptors. Therefore, a 15-min infusion of BQ-123 (1 microM) prevented the vasoconstrictor response to ET-1 (10 nM). Sixty minutes after the end of the infusion, the vasoconstrictor response to ET-1 was fully restored to control levels. Conversely, when the selective ETB receptor antagonist was administered, the vasoconstrictor response to ET-1 was markedly potentiated. After the infusion of the antagonist was ended there was no further potentiation of the response to ET-1. Co-infusion of supramaximal concentrations of BQ-123 (1 microM) and BQ-788 (10 nM) still reduced by about 90% the vasoconstrictor response to ET-1. However, after removal of the antagonists, the vasoconstriction to ET-1 was potentiated by more than 75% compared to control (n = 8; p < 0.05). Similarly, a mixture of both antagonists was less effective than BQ-123 alone in reducing the pressor effect of ET-1 in anesthetized rabbits. These results show that blockade of ETB receptors produces marked potentiation in the vasoconstrictor responses to ET-1. Furthermore, a rebound hyperresponsiveness is found after treatment with a mixture of BQ-123 and BQ-788. These results suggest that the systemic and renal vasculatures would respond to ET-1 with enhanced vasoconstriction after treatment with mixed ETA/ETB receptor antagonists owing to the slow reversibility of the antagonism of the ETB receptor-mediated release of nitric oxide.

Characterization of endothelin receptors and endothelin-converting enzyme activity in the rabbit lung.

We had earlier shown that endothelin-1 (ET-1)-induced vasoconstriction and prostacyclin (PGI2) release in the rabbit kidney are exclusively linked to ETA receptor activation. In contrast, another study showed that ET-1 inhibited platelet aggregation ex vivo through the release of PGI2 solely via the activation of ETB receptors. In an attempt to identify the organs involved in the ET-1-induced release of PGI2 in vivo, we characterized the receptors responsible for the release of this eicosanoid and also monitored the activity of the endothelin-converting enzyme (ECE) in rabbit pulmonary lobe. In this perfused organ, a 5-min infusion of ET-1 (50 nM) triggered a marked release of PGI2 and an increase in perfusion pressure, both of which were mimicked by the selective ETB agonist IRL 1620 (0.5 microM). Furthermore, a selective ETB antagonist, BQ-788 (10 nM), markedly blunted the release of PGI2 induced by ET-1 (50 nM). On the other hand, ET-2 was also able to trigger the release of PGI2 and to increase the perfusion pressure in this organ. The immediate precursor of ET-1, big ET-1 (0.5 microM), also induced a protracted increase in perfusion pressure. In contrast, big ET-2 (0.5 microM) was significantly weaker than big ET-1 in increasing perfusion pressure. Our results suggest that the receptors responsible for the release of PGI2 in the lung are (in contrast to the kidney) predominantly of the ETB type. Furthermore, the ECE localized in the rabbit pulmonary vasculature is relatively selective for big ET-1. On the basis of these results, we suggest that ETB-mediated inhibition of platelet aggregation ex vivo is due to a marked release of PGI2 generated from the pulmonary vasculature in the rabbit.