Pharmacological knockout of endothelin ET(A) receptors

We employed newly developed antagonists, which are specific for endothelin ET(A) receptors, to test whether this drug could mimic the phenotype of the mouse with corresponding gene knock out. Newborn rats, whose dams were given the ET(A) antagonist from day 7 of gestation, exhibited the typical ET(A)-lacking phenotypes like craniofacial abnormalities and major vessel anomalies. Interestingly, craniofacial abnormality was seen in the pups that were exposed to the drug in the mid-gestational period, while another phenotype, patent ductus arteriosus (DA), was seen in the pups that were exposed to the drug in the late gestation.We have focused on the function of the ET system in DA closure after birth because the animals with a genetic defect of ET(A) would die of suffocation shortly after birth. Rat pups were delivered by Caesarean section and were given the antagonist intraperitoneally. The antagonists caused an inhibition of DA closure in vivo at 3 h after birth when DA closure was completed in the control pups. Next, we tested the potential utilities of the ET(A) specific antagonists in tocolysis with NSAIDs which sometimes leads to a closure of fetal DA in utero. Indomethacin administration to rat dams resulted in the constriction of DA in utero which was cancelled by the co-administration of the antagonists. These results suggested that ET(A) plays a physiological role in the postnatal closure of the rat DA in vivo and that ET(A) specific antagonists may be able to leave fetal DA intact during tocolysis with NSAIDs.     

Blockade of endothelin ET(A)/ET(B) receptors favors a role for endothelin during acute Trypanosoma cruzi infection in rats

Endothelin has been implicated in the pathogenesis of experimental and human Chagas' disease (American trypanosomiasis). In the present study, we tested the effect of bosentan, an antagonist of both ET(A) and ET(B) endothelin receptors, on parasitemia, histopathology (heart and diaphragm), heart levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, interferon (IFN)-gamma, CCL2, CCL3 and CCL5, and the serum levels of nitrate/nitrite (NOx). Bosentan treatment was accompanied by a significant increase in parasitemia and tissue parasitism or inflammation. In vehicle-treated rats, Trypanosoma cruzi infection increased the cardiac levels of TNF-alpha, IFN-gamma and IL-10, at day 9 post inoculation, and the TNF-alpha remained elevated until day 13. The infection also caused a significant increase in the cardiac levels of the chemokines CCL2 (9, 13 and 18 days) and CCL3 (13 and 18 days). Bosentan-treatment had no significant effect on the infection-associated increase in IFN-gamma and chemokine concentrations. There was a lower increase in IL-10 at day 9 and this was mirrored by a greater increase of TNF-alpha at day 13, in comparison with vehicle-treated rats. These latter findings correlated well with the enhanced inflammatory process in hearts of bosentan-treated infected rats. Bosentan treatment reduced the infection-associated increase in NOx serum concentration. Altogether, our data suggest that ET action on ET(A) and ET(B) receptors may play a role in the initial control of T. cruzi infection in rats probably by interfering in NO production.     

Quantification of endothelin receptor subtypes in peripheral tissues reveals downregulation of ET(A) receptors in ET(B)-deficient mice

We have previously shown that in homozygous endothelin (ET)(B)-/- deficient mice, ET(A) receptor density is significantly downregulated in the brain by 45%. In these mice, plasma ET-1 levels are elevated. Our aim was to use quantitative autoradiography to establish the distribution of ET receptor subtypes in peripheral tissues from wild-type mice and to measure the density of the ET(A) subtype in ET(B)-/- knockout animals. Our second aim was to test whether deletion of ET(B) receptors, which is associated with elevated plasma levels of ET-1, would also reduce ET(A) expression in the periphery. In longitudinal sections from wild-type mice, the highest densities of ET(A) receptors localized to major organs including the ventricle of the heart, lung, and liver parenchyma. High densities of ET(A) receptors were detected in the smooth muscle layer of the vasculature such as intrarenal vessels as well as the smooth muscle layer and epithelial cells of the gastrointestinal tract. In these tissues, the ET(A) subtype was more abundant, representing between 60% and 100% of the ET receptors. ET(B) receptors predominated in the medulla of kidney, with high densities also localizing to glomeruli within the cortex and to the sinusoids from the liver. Lower densities of ET(B) receptors were also present in the lung, heart, liver, and the smooth muscle layer of the gastrointestinal tract. In ET(B)-/- knockout mice, ET(B) receptors were not detected as expected by either ligand binding or immunocytochemistry. The pattern of ET(A) receptor distribution in the ET(B)-/- knockout mice was similar to the controls, but the density of ET(A) receptors was significantly reduced in the lung by 39%. Diminished responses to the endogenous agonist after repeated stimulation are an important feature of G-protein signaling, preventing potential damage to the overstimulated cell, and it is likely that downregulation occurs in response to higher circulating levels of ET-1.     

Endothelin ET(A) and ET(B) receptors in postnatal intestine

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with (125)I-labeled ET-1 in membranes from endothelium-denuded (E(-)) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive (125)I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E(+)) and E(-) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ET(A) receptor antagonist BQ-123, competitive (125)I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E(+) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ET(B) receptor.     

Benzofuro[3,2-b]pyridines as mixed ET(A)/ET(B) and selective ET(B) endothelin receptor antagonists

The discovery, synthesis and structure-activity relationships of a series of novel benzofuro[3,2-b]pyridines as non-selective endothelin ET(A)/ET(B) as well as selective ET(B) receptor antagonists are described. The most potent non-selective inhibitor 7s displayed an IC50 of 21 nM and 41 nM for ET(A) and ET(B) receptors, respectively, whereas 7ee merely showed affinity for the ET(B) receptor (IC50 = 3.6 nM).     

Central endothelin ET(B) receptors mediate IL-1-dependent fever induced by preformed pyrogenic factor and corticotropin-releasing factor in the rat

Blockade of central endothelin ET(B) receptors inhibits fever induced by LPS in conscious rats. The contribution of ET(B) receptor-mediated mechanisms to fever triggered by intracerebroventricular IL-6, PGE2, PGF(2alpha), corticotropin-releasing factor (CRF), and preformed pyrogenic factor derived from LPS-stimulated macrophages (PFPF) was examined. The influence of natural IL-1 receptor antagonist or soluble TNF receptor I on endothelin (ET)-1-induced fever was also assessed. The selective ET(B) receptor antagonist BQ-788 (3 pmol icv) abolished fever induced by intracerebroventricular ET-1 (1 pmol) or PFPF (200 ng) and reduced that caused by ICV CRF (1 nmol) but not by IL-6 (14.6 pmol), PGE2 (1.4 nmol), or PGF(2alpha) (2 nmol). CRF-induced fever was also attenuated by bosentan (dual ET(A)/ET(B) receptor antagonist; 10 mg/kg iv) but unaffected by BQ-123 (selective ET(A) receptor antagonist; 3 pmol icv). alpha-Helical CRF(9-41) (dual CRF1/CRF2 receptor antagonist; 6.5 nmol icv) attenuated fever induced by CRF but not by ET-1. Human IL-1 receptor antagonist (9.1 pmol) markedly reduced fever to IL-1beta (180 fmol) or ET-1 and attenuated that caused by PFPF or CRF. Murine soluble TNF receptor I (23.8 pmol) reduced fever to TNF-alpha (14.7 pmol) but not to ET-1. The results of the present study suggest that PFPF and CRF recruit the brain ET system to cause ET(B) receptor-mediated IL-1-dependent fever.     

Hypertension induced by blockade of ET(B) receptors in conscious nonhuman primates: role of ET(A) receptors

The role of endothelin-B (ET(B)) receptors in circulatory homeostasis is ambiguous, reflecting vasodilator and constrictor effects ascribed to the receptor and diuretic and natriuretic responses that could oppose the hypertensive effects of ET excess. With the use of conscious, telemetry-instrumented cynomolgus monkeys, we characterized the hypertension produced by ET(B) blockade and the role of ET(A) receptors in mediating this response. Mean arterial pressure (MAP) and heart rate (HR) were measured 24 h/day for 24 days under control conditions and during administration of the ET(B)-selective antagonist A-192621 (0.1, 1.0, and 10 mg/kg bid, 4 days/dose) followed by coadministration of the ET(A) antagonist atrasentan (5 mg/kg bid) + A-192621 (10 mg/kg bid) for another 4 days. High-dose ET(B) blockade increased MAP from 79 +/- 3 (control) to 87 +/- 3 and 89 +/- 3 mmHg on the first and fourth day, respectively; HR was unchanged, and plasma ET-1 concentration increased from 2.1 +/- 0.3 pg/ml (control) to 7.24 +/- 0.99 and 11.03 +/- 2.37 pg/ml. Atrasentan + A-192621 (10 mg/kg) decreased MAP from hypertensive levels (89 +/- 3) to 75 +/- 2 and 71 +/- 4 mmHg on the first and fourth day, respectively; plasma ET-1 and HR increased to 26.64 +/- 3.72 and 28.65 +/- 2.89 pg/ml and 113 +/- 5 (control) to 132 +/- 5 and 133 +/- 7 beats/min. Thus systemic ET(B) blockade produces a sustained hypertension in conscious nonhuman primates, which is mediated by ET(A) receptors. These data suggest an importance clearance function for ET(B) receptors, one that influences arterial pressure homeostasis indirectly by reducing plasma ET-1 levels and minimizing ET(A) activation.     

Cellular localization of the endothelin receptor subtypes ET(A) and ET(B) in the rat heart and their differential expression in coronary arteries,veins, and capillaries

In the heart, the endothelin (ET)/endothelin-receptor system is markedly involved in pathophysiological mechanisms underlying various cardiac diseases. Based upon pharmacological studies both ET-receptor subtypes take part in the regulation of coronary vascular tone, however, their detailed cellular distribution in the coronary vascular bed based upon direct mRNA and protein detection is unknown. This issue was addressed in the rat heart by means of non-radioactive in situ hybridization, RT-PCR, and immunohistochemistry. Expression of vascular ET(A)-receptors was detected in arterial smooth muscle and capillary endothelium while ET(B)-receptors were present in arterial, venous, and capillary endothelium, and in arterial and venous smooth muscle cells. This differential distribution of the ET-receptor subtypes supports the concept that ET(A)- as well as ET(B)-receptors mediate arterial vasoconstriction, while postcapillary vascular resistance is exclusively regulated by ET(B)-receptors. The observed capillary endothelial expression of the ET(A)-receptor correlates with the known ability of ET(A)-receptor antagonists to attenuate increases in cardiac microvascular permeability during endotoxin shock and ischemia/reperfusion injury.     

Possible bi-directional link between ET(A) receptors and protein kinase C in rat blood vessels

Possible links have been investigated between activation of protein kinase C (PKC) and endothelin (ET) production by small blood vessels. Perfusion pressures were recorded from rat isolated mesenteric artery, with or without the small intestine attached, before and after addition to the perfusate of either ET-1, ET-3 or the PKC activator 12-deoxyphorbol 13-phenylacetate (DOPPA). Rises in perfusion pressure in response to ET-1 (10(-8) M)or DOPPA (10(-6) M) were reduced significantly by pre-treatment with either the ET(A) receptor antagonist PD151242 (10(-6) M) or the PKC inhibitor Ro 31-8220 (10(-6) M). ET-3 (10(-8) M) had a significant, albeit small, effect only when the gut was still attached to the mesentery. Inthis latter preparation ET-1 and DOPPA increased the permeability of villi microvessels to colloidal carbon in the perfusate. This effect of DOPPA was reduced by pre-treatment with either PD151242 or Ro 31-8220, but the effects of ET-1 were reduced significantly only by Ro 31-8220. ET-3 (10(-8) M) was without effect. The results suggest a possible bi-directional link between ET(A) receptors and PKC in the intestinal vasculature.     

Differential trafficking and desensitization of human ET(A) and ET(B) receptors expressed in HEK 293 cells

Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ET(A) and ET(B) receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ET(A) and ET(B) receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ET(A) receptors, 83 +/- 2% of these receptors colocalized with pan cadherin. In ET(B) receptor-transfected cells, 54 +/- 2% of the receptor colocalized with pan cadherin. When ET(A) and ET(B) receptors were cotransfected, 97 +/- 1% of ET(B) receptors colocalized with ET(A) receptors and 84 +/- 2% of ET(B) receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ET(B) receptor agonist) increased [Ca2+]i in cells transfected with ET(A) or ET(B) receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ET(B) receptors desensitized faster (t(1/2) = 21 +/- 1 sec) than ET(A) receptors (t(1/2) = 48 +/- 1 sec). S6c-induced increases in [Ca2+]i desensitized in cells expressing ET(B) receptors only (t(1/2) = 17 +/- 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ET(A) receptors localize to the cell membrane, whereas ET(B) receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ET(B) receptors desensitize faster than ET(A) receptors, but receptor coexpression eliminates desensitization. Finally, ET(A) and ET(B) receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.     

Endothelin ET(A) but not ET(B) receptors mediate contraction of common bile duct

Endothelin (ET) causes contraction of the gallbladder. To investigate effects of ET in the common bile duct, we measured contraction of longitudinal muscle strips from guinea pig common bile ducts induced by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the common bile duct. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. ET-1 caused tetrodotoxin and atropine-insensitive contraction. In terms of maximal tension of contraction, ET-1, ET-2 and ET-3 were equal in efficacy. However, sarafotoxin S6c, a selective ET(B) receptor agonist, caused only a negligible contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. The ET-1-induced contraction was inhibited by BQ-123, an ET(A)-receptor-selective antagonist, but not by BQ-788, an ET(B)-receptor-selective antagonist. In addition, the combination of both antagonists, BQ-123 and BQ-788, inhibited ET-1 induced contraction but did not potentiate the inhibition caused by BQ-123 alone. These indicate that ET(A) but not ET(B) receptors mediate the contraction. Autoradiography localized 125I-ET-1 binding to the smooth muscle layer. Binding of 125I-ET-1 to the smooth muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves indicated the presence of ET(A) and ET(B) receptors. These results demonstrate that ET causes contraction of longitudinal muscle of the common bile duct. Different from the gallbladder, which possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction, the common bile duct possesses two classes of ET receptors, but only the ET(A) receptor mediates the contraction.     

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with ¹²⁵ I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K_d values of 1 × 10^?10 M and 5 × 10^?9 M. The numbers of high- and low- affinity receptors were 1 × 10⁴ and 2 × 10⁵ per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.     

Imaging endothelin ET(B) receptors using [18F]-BQ3020: in vitro characterization and positron emission tomography (microPET)

The endothelin (ET) receptor system has been shown to play a role in a number of vascular diseases. We have synthesized 18F-and 11C-labeled radioligands to enable in vivo imaging of the fundamental processes involved in ET receptor pharmacology in normal and diseased tissue using positron emission tomography (PET). One aim is to elucidate the proposed role of the ET(B) subtype as clearing receptor, removing ET-1 from the circulation, and whether this is an important mechanism to limit the detrimental effects caused by upregulated ET-1 in disease. To image ET(B) receptors we have labeled the selective agonist BQ3020 with 18F. In vitro characterization verified that [18F]-BQ3020 bound with a single subnanomolar affinity (K(D) = 0.34 +/- 0.10 nM, B(max) = 9.23 +/- 3.70 fmol/mg protein) to human left ventricle. Binding of [18F]-BQ3020 to human kidney was inhibited by ET-1 and unlabeled BQ3020 but not by the ET(A) selective antagonist FR139317, confirming that selectivity for the ET(B) receptor was retained. In vitro autoradiography revealed, as expected, high levels of ET(B) receptor densities in lung and kidney medulla, whereas kidney cortex and heart showed lower levels of ET(B) receptor densities. Furthermore, a high level of [18F]-BQ3020 binding was found to colocalize to macrophages in atherosclerotic coronary arteries. MicroPET studies demonstrated high uptake of [18F]-BQ3020 in ET(B) receptor-rich tissue, including lung, liver and kidney. The in vivo biodistribution of [18F]-BQ3020 was comparable to that previously obtained for [18F]-ET-1, supporting our hypothesis that the ET(B) receptor plays a significant role in the uptake of ET-1. In conclusion, [18F]-BQ3020 has retained high affinity and selectivity, allowing imaging of ET(B) receptor distributions in vitro and in vivo in human and animal tissue. Furthermore, in vitro data suggest that [18F]-BQ3020 potentially can be used to image atherosclerotic lesions in vivo using PET.     

Endothelin-I induces gene expression through stimulation of endothelin type a receptors in normal rat kidney cells

RNA blots of total cellular RNA isolated from quiescent and endothelin (ET-1)-stimulated normal rat kidney (NRK) cells demonstrated that ET-1 induced the expression of c-jun, jun B, and c-fos mRNA in a time-dependent manner with maximal expression of mRNA by 1 hr after the addition of ET-1. Five hundred picomolal ET-1 was sufficient to induce maximal mRNA expression. These data agreed with saturation experiments which demonstrated that maximal binding of [¹²⁵I]ET-1 was achieved at concentrations greater than 100 pM. The K_d and B_max values for [¹²⁵I]ET-1 binding to NRK membranes were 20.5 pM and 22.2 fmol/mg protein, respectively. Competition experiments for the binding of [¹²⁵I]ET-1 to NRK membranes demonstrated that ET-1 was a more potent inhibitor (K_i = 0.047 nM) than ET-3 (K_i = 10.8 nM). No specific binding of [¹²⁵I]ET-3 (40 or 500 pM) to NRK membranes could be observed. The expression of c-jun, jun B, and c-fos mRNA was inhibited by the endothelin type A receptor (ET)-selective antagonist, BQ-123. Thus, these data demonstrate that ET-1 mediates the expression of immediate response gene mRNA in NRK cells via the ET_A receptor. ET-1 stimulation of NRK cells also upregulated EGF receptors, providing a possible mechanism for ET-1 complementation of epidermal growth factor (EGF) mitogenicity in NRK cells. © 1995 Wiley-Liss, Inc.     

Ontogeny of endothelin and its receptors in rat brain.

The ontogeny of endothelin (ET) system in rats was studied in preterm (18 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (Kd) and density (Bmax) were determined using [125I] ET-1 and [125I] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 +/- 3.18 pg/g protein in preterm, 47.37 +/- 5.31 pg/g protein in term and 48.30 +/- 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [125I] ET-1 and [125I] SRT 6b. The Kd values for [125I] ET-1 and [125I] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [125I] ET-1 and [125I] SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bmax values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density.     

Role of ET(A) receptors in experimental ANG II-induced hypertension in rats

The objectives were to determine if ANG II-induced hypertension is maintained by activation of endothelin type A (ET(A)) receptors by endogenous ET-1 and if this effect is influenced by salt intake. Male rats were maintained on high sodium intake (HS; 6 meq/day) or on normal sodium intake (NS; 2 meq/day). Hypertension was produced by intravenous infusion of ANG II (5 ng/min) for 15 days. Five-day oral dosing with the selective ET(A)-receptor antagonist ABT-627 (~2 mg. kg(-1). day(-1)) reduced mean arterial pressure (MAP) to baseline levels in rats on HS receiving ANG II infusion, but it did not affect MAP in normotensive HS controls. In rats on NS, ABT-627 only transiently decreased MAP in rats receiving ANG II and slightly reduced MAP in normotensive controls. ABT-627 produced mild retention of sodium and water in NS rats receiving ANG II, but not in any other group. These results indicate that ET-1 plays a role in ANG II-induced hypertension via activation of ET(A) receptors and that this role is more prominent in rats on HS.