The interaction of endothelin receptor responses in the isolated perfused rat lung

To gain more insight into the complex pulmonary interactions of endothelins (ET), we studied airway and vascular responses to endothelins in isolated perfused rat lungs in the presence of the novel ET_B-receptor antagonist BQ788. In particular we focused on airway responses and on prostacyclin release. The effectiveness of BQ788 in our system was shown by its ability to concentration-dependently prevent vasoconstriction (IC₅₀ 0.1μM), bronchoconstriction (IC₅₀ 0.1μM) and prostacyclin production (IC₅₀<0.1μM) induced by the ET_B-receptor agonist IRL1620 (1nmol). Airway responses to ET-1: ET-1-induced bronchoconstriction was aggravated by BQ123 (1 or 8μM), while BQ788 pretreatment (1 or 8μM) showed no significant effect. Simultaneous treatment with 8μM BQ123 and BQ788 attenuated the ET-1-induced bronchoconstriction. Vascular responses to ET-1: ET-1 (1nmol)-induced vasoconstriction was potentiated by BQ788 (1 or 8μM), but attenuated by the ET_A-receptor antagonist BQ123 (1μM). In the presence of BQ788 diminished amounts of the stable prostacyclin metabolite 6-keto-PGF_1α were detected in the perfusate. Simultaneous treatment with 8μM BQ123 and BQ788 completely prevented the ET-1-induced vasoconstriction. Conclusions: Both ET_A- and ET_B-receptors contribute to ET-1-induced vasoconstriction and bronchoconstriction. The ET-1-induced vasoconstriction is attenuated by stimulation of ET_B-receptors, a response that is partly mediated by prostacyclin. Due to the mutual interactions between ET_A- and ET_B-receptors, simultaneous inhibition of both receptors is required to prevent the deleterious effects of ET-1 on lung functions. Received: 17 October 1996 / Accepted: 16 May 1997     

Analysis of ET‐A and ET‐B receptors using an isolated perfused rat lung preparation

Aims and Methods: The pulmonary and vascular effects of endothelin‐1 receptor activation were studied in isolated perfused and ventilated lung preparations from rat. The responses to endothelin‐1 (ET‐1) and the endothelin B (ET_B) receptor agonist sarafotoxin 6c (S6c) were characterized using the endothelin A (ET_A)‐receptor antagonist FR 139317, the ET_B‐receptor antagonist BQ 788 and the combined ET_A/ET_B‐receptor antagonist Bosentan. The respiratory parameter airway conductance (G_aw) and the vascular parameter perfusion flow were analysed simultaneously. Results: Concentration–response curves for ET‐1 administered intra‐arterially revealed that its most potent effect was on the vascular side while S6c had a more potent effect on airway conductance. ET‐1, given as a bolus dose intra‐arterially (100 μL of 0.2 nm ), induced a strong‐ and long‐lasting contraction of the vasculature while only a less pronounced contraction was seen in the airways. Neither of the antagonists had a significant effect per se on G_aw or perfusion flow. FR 139317 reduced the effect of ET‐1 on perfusion flow by about 50%, while airway conductance was augmented. BQ 788 enhanced the decrease in perfusion flow by ET‐1 while G_aw was not influenced. The combined ET_A/ET_B antagonist Bosentan powerfully prevented the ET‐1‐induced decrease in G_aw but did not alter its reduction in perfusion flow. Conclusions: The potent effect of ET‐1 on the vascular side of the lung is mediated mainly through ET_A receptors, whereas both ET_A and ET_B receptors are involved in G_aw in the rat lung.     

Intrastriatal injection of endothelin evokes dopaminergic turning behaviour in rats through activation of the ETB receptor

Contralateral intrastriatal injection of 0.1 pmol or 1 pmol of endothelin-1 produced ipsilateral turning behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway. This effect could be abolished by pretreatment with either the endothelinET_A/B receptor antagonist bosentan (1 nmol, intrastriatally) or the dopamine D₂ receptor antagonist raclopride (0.1 mg/kg, s.c.) suggesting that endothelin is acting at endothelin receptors to evoke ipsilateral turning behaviour and that this response is mediated by dopamine. Similar ipsilateral turning behaviour was observed upon intrastriatal injection of 1 pmol of endothelin-3 or the specific ET_B receptor agonist, [Ala^1,3,11,15]endothelin-1 when compared to endothelin-1. Pretreatment with the specific ET_B receptor antagonist BQ788 blocked the ipsilateral turning response to intrastriatal injection of endothelin-1 while pretreatment with the specific ET_A receptor antagonist BQ123 did not significantly change the response to injection of endothelin-1. This indicates that endothelin-1, which has affinity for both ET_A and ET_B receptors, is most likely acting at the ET_B receptor to elicit its effect. These results suggest that low doses of endothelin may act at ET_B receptors to evoke the release of dopamine from the striatum in vivo.     

Expression of the human multidrug resistance gene mdr1 in leukemic cells and its application in studying P-glycoprotein antagonists

Objective To explore the role of endothelin (ET) in the pathogenesis of exercise-induced asthma (EIA), we investigated the effects of ET(B) receptor antagonists, ET-1 (11-21)fragment and N-cis-2,6-dimethylpi-peridinocardonyl-L-γ-methylleucyl-D-1-methoxycarbonyl tryptophanyl-D-norleucine (BQ788) on broncho-constriction elicited by isocapnic hyperpnea in guinea pigs. Methods Eighteen pathogen-free Hartley guinea pigs were randomly divided into three groups. A: normal saline (NS) inhalation control group (n=6), B: BQ788 group (n=6), and C: ET-1(11-21) fragment group (n=6). Guinea pigs were anesthetized with pentobarbital sodium. After measuring the basal value of lung resistance (R[L]) and dynamic compliance of the respiratory system (Cdyn), NS (0.96 ml), BQ788 (9 nmol) and ET-1(11-21)fragment (9 nmol) were inhaled. A rodent respirator with a dry 5%CO(2)-95%O(2) mixture at room temperature provided mechanical ventilation (V[T] 8 ml/animal, 100 breaths/min) for 5 min. R[L] and Cdyn of the 3 groups were measured again after isocapnic hyperpnea challenge. Results In the control group, isocapnic hyperpnea of dry gas elicited a marked increase in R[L] and decrease in Cdyn. R[L] and Cdyn of the guinea pigs from BQ788 group and ET-1(11-21)fragment group did not change significantly. Conclusion It was demonstrated that selective ET(B) receptor antagonists, ET-1(11-21) fragment and BQ788, inhibited the bronchoconstriction induced by isocapnic hyperpnea in guinea pigs. The data showed that ETs are potent constrictors of guinea pig airway smooth muscle via a direct effect on ET receptors. It was suggested that ET receptor antagonists, especially ET(B) receptor antagonist, might be beneficial in preventing EIA.     

BQ‐788, A Selective Endothelin ETB Receptor Antagonist

We describe characteristics of a selective endothelin (ET) ET_B receptor antagonist, BQ‐788 [N‐cis‐2,6‐dimethylpiperidinocarbonyl‐L‐γ‐methylleucyl‐D‐1‐methoxycarbonyltryptophanyl‐D‐norleucine], which is widely used to demonstrate the role of endogenous or exogenous ETs in vitro and in vivo. In vitro, BQ‐788 potently and competitively inhibited ¹²⁵I‐labeled ET‐1 binding to ET_B receptors in human Girrardi heart cells (hGH) with an IC₅₀ of 1.2 nM, but only poorly inhibited the binding to ET_A receptors in human neuroblastoma cell line SK‐N‐MC cells (IC₅₀, 1300 nM). In isolated rabbit pulmonary arteries, BQ‐788 showed no agonistic activity up to 10 μ and competitively inhibited the vasoconstriction induced by an ET_B‐selective agonist (pA₂, 8.4). BQ‐788 also inhibited several bioactivities of ET‐1, such as bronchoconstriction, cell proliferation, and clearance of perfused ET‐1. Thus, it is confirmed that BQ‐788 is a potent, selective ET_B receptor antagonist. In vivo, in conscious rats, BQ‐788, 3 mg/kg/h, i.v., completely inhibited a pharmacological dose of ET‐1‐ or sarafotoxin6c (S6c) (0.5 nmol/kg, i.v.)‐induced ET_B receptor‐mediated depressor, but not pressor responses. Furthermore, BQ‐788 markedly increased the plasma concentration of ET‐1, which is considered an index of potential ET_B receptor blockade in vivo. In Dahl salt‐sensitive hypertensive (DS) rats, BQ‐788, 3 mg/kg/h, i.v., increased blood pressure by about 20 mm Hg. It is reported that BQ‐788 also inhibited ET‐1‐induced bronchoconstriction, tumor growth and lipopolysaccharide‐induced organ failure. These data suggest that BQ‐788 is a good tool for demonstrating the role of ET‐1 and ET_B receptor subtypes in physiological and/or pathophysiological conditions.     

BQ123对体外循环缺血再灌注心肌胰岛素抵抗干预作用的实验研究

目的:探讨内皮素受体拮抗剂BQ123对体外循环缺血再灌注心肌胰岛素抵抗(IR)的干预作用。方法:将18条杂种犬分为3组(n=6),Ⅰ组主动脉阻断30min,Ⅱ组主动脉阻断120min,Ⅲ组主动脉阻断120min且心脏停博液中加入BQ123。分别于体外循环转流前、主动脉开放后第15、45、75min4个时点,采集动脉、冠状静脉窦血液标本并进行右房心肌活检,测定各相关指标。结果:与Ⅱ组比较,Ⅲ组的动脉血糖、胰岛素、IR指数、应急激素、血浆内皮素-1及血浆肿瘤坏死因子-α水平升高幅度较小,心肌对葡萄糖的摄取和利用提前至再灌注45min,恢复至正常值更快(P<0.05或P<0.01)。结论:BQ123可以减轻体外循环缺血再灌注过程中的心肌IR,对其引起的损伤具有保护作用。     

ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]ET-1 in a biphasic manner, in contrast to an ET(B)-selective agonist, IRL-1620, that was ineffective. The ET(B)-selective antagonist, BQ-788, inhibited [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET(A) and -ET(B) antibodies confirmed a predominant expression of the ET(A) receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca(2+) resulted in a shift to the right of the ET-1 concentration-response curve. Both the L-type voltage-operated Ca(2+) channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET(A) receptors are expressed and functionally coupled to rise of [Ca(2+)](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca(2+)](i) increase is triggered by Ca(2+) release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca(2+) channels and ryanodine receptors participate in sustaining the Ca(2+) response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca(2+)](i) increase.     

内皮素及其A受体在兔颅脑创伤后脑血流量变化中的作用及意义

目的:了解内皮素(ET)及其A受体在颅脑创伤后脑血流量变化中的作用及意义.方法:130只家兔致以左额顶叶为着力部位的加速性撞击伤,动态观察脑组织内皮素含量变化、内皮素A受体mRNA表达和定位变化,内皮素A受体拮抗剂BQ123的作用及上述变化对脑血流量变化的影响.结果:脑创伤后血浆、脑脊液和局部脑组织内皮素含量显著升高;内皮素A受体mRNA主要分布于脑血管内皮细胞,表达显著增强;创伤后局部脑血流量显著降低,应用BQ123治疗的动物,血流量下降被明显抑制.结论:内皮素通过其A受体的调节在颅脑创伤后脑缺血的形成中有重要作用;应用内皮素A受体拮抗剂可以防治颅脑创伤后脑缺血.     

19F NMR in vivo spectroscopy reflects the effectiveness of perfusion-enhancing vascular modifiers for improving gemcitabine chemotherapy.

Nuclear magnetic resonance spectroscopy of fluorine-19 ((19)F NMR) has proven useful for evaluating kinetics of fluorinated chemotherapy drugs in tumors in vivo. This work investigated how three perfusion-enhancing vascular modifiers (BQ123, thalidomide, and Botulinum neurotoxin type A [BoNT-A]) would affect the chemotherapeutic efficacy of gemcitabine, a fluorinated drug widely used in human cancer treatment. Murine tumor growth experiments demonstrated that only BoNT-A showed a strong trend to enhance tumor growth inhibition by gemcitabine (1.7 days growth delay, P = 0.052, Student t-test). In accord with these results, (19)F NMR experiments showed that only BoNT-A increased significantly the uptake of gemcitabine in tumors (50% increase, P = 0.0008, Student t-test). Further experiments on gemcitabine kinetics (NMR vs time) and distribution ((19)F MRI) confirmed the uptake-enhancing properties of BoNT-A. The results of this study demonstrate that (19)F NMR can monitor modulation of the pharmacokinetics of fluorinated chemotherapy drugs in tumors. The results also show that (19)F NMR data can give a strong indication of the effectiveness of perfusion-enhancing vascular modifiers for improving gemcitabine chemotherapy in murine tumors. (19)F NMR is a promising tool for preclinical evaluation of such vascular modifiers and may ultimately be used in the clinic to monitor how these modifiers affect chemotherapy.