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It is unclear how hepatic glucocorticoid receptor (GR) function and hypothalamic-pituitary-adrenal axis tone contribute to the diabetic state and in particular whole-body glucose fluxes. We have previously demonstrated that long-term exposure to hepatic GR inhibition lowers glucose levels in ob/ob mice (J Pharmacol Exp Ther 2005;314:191). The purpose of this study was to determine the effects of a novel GR antagonist (A-348441) on whole-body glucose fluxes in a model of insulin resistance, the Zucker fatty (fa/fa) rat. After an overnight fast, euglycemic-hyperinsulinemic clamp studies were performed 2 hours after single oral dosing as follows: (1) A-348441 at 100 mg/kg or (2) vehicle. Furthermore, effects of 1 week of treatment with either vehicle or A-348441 (3, 10, 30, or 100 mg/kg PO, once per day) were investigated in separate groups of rats fasted overnight and given a final dose of their respective compound, followed 2 hours later by a euglycemic-hyperinsulinemic clamp. One week after catheter implantation, body weight returned to presurgery levels, with no difference between groups. A single, 100-mg/kg dose of A-348441 significantly increased glucose infusion rate 4-fold (P < .05) and reduced endogenous glucose production by 37% (P < .05) but did not change glucose disposal. After 1 week of sub–long-term dosing, fasting glucose levels were reduced dose-dependently with A-348441 vs vehicle (−8%, not significant; −14%, −20%, and −25%, P < .05, at 3, 10, 30, and 100 mg/kg, respectively) with no observed hypoglycemia or change in fasting insulin levels. A-348441 increased the glucose infusion rates after 1-week treatment by 1.3-, 5.7-, 7.3-, and 6.4-fold (P < .05). Endogenous glucose production was decreased (−25%, −44%, −50%, and −61%, P < .05), whereas glucose disposal was increased (29% and 13%, not significant; 23% and 34%, P < .05), with A-348441. In summary, single-dose treatment with the liver-selective GR antagonist A-348441 decreases glucose production with no effect on glucose disposal or fasting glucose levels. After 1 week of treatment with A-348441, (1) there was no effect on body weight, (2) fasting glucose levels decreased, (3) both glucose disposal and glucose infusion rate increased during clamping, and (4) endogenous glucose production was greatly reduced. In addition, hepatic glucose production was highly correlated with fasting glucose levels (r = 0.97). In conclusion, these results indicate that A-348441 increases insulin sensitivity at both the liver and peripheral tissues, leading toward a normalization of the insulin resistant state. Furthermore, with 1-week vs single-dose liver-selective glucocorticoid antagonism, we have determined that the peripheral effect is secondary to the primary event of reduced hepatic glucose production. The approach of inhibiting the hepatic GR may be an advantageous treatment paradigm for individuals with type 2 diabetes mellitus.

The net contribution of endothelin type A (ETA) and type B (ETB) receptors in blood pressure regulation in humans and experimental animals, including the conscious mouse, remains undefined. Thus we assessed the role of ETA and ETB receptors in the control of basal blood pressure and also the role of ETA receptors in maintaining the hypertensive effects of systemic ETB blockade in telemetry-instrumented mice. Mean arterial pressure (MAP) and heart rate were recorded continuously from the carotid artery and daily (24 h) values determined. At baseline, MAP ranged from 99 ± 1 to 101 ± 1 mmHg and heart rate ranged between 547 ± 15 and 567 ± 19 beats/min ( n = 6). Daily oral administration of the ETB selective antagonist A-192621 [10 mg/kg twice daily] increased MAP to 108 ± 1 and 112 ± 2 mmHg on days 1 and 5, respectively. Subsequent coadministration of the ETA selective antagonist atrasentan (5 mg/kg twice daily) in conjunction with A-192621 (10 mg/kg twice daily) decreased MAP to baseline values on day 6 (99 ± 2 mmHg) and to below baseline on day 8 (89 ± 3 mmHg). In a separate group of mice ( n = 6) in which the treatment was reversed, systemic blockade of ETB receptors produced no hypertension in animals pretreated with atrasentan, underscoring the importance of ETA receptors to maintain the hypertension produced by ETB blockade. In a third group of mice ( n = 10), ETA blockade alone (atrasentan; 5 mg/kg twice daily) produced an immediate and sustained decrease in MAP to values below baseline (baseline values = 101 ± 2 to 103 ± 2 mmHg; atrasentan decreased pressure to 95 ± 2 mmHg). Thus these data suggest that ETA and ETB receptors play a physiologically relevant role in the regulation of basal blood pressure in normal, conscious mice. Furthermore, systemic ETB receptor blockade produces sustained hypertension in conscious telemetry-instrumented mice that is absent in mice pretreated with an ETA antagonist, suggesting that ETA receptors maintain the hypertension produced by ETB blockade.

A rat model of acute pulmonary air embolism (APAE) was developed. These animals had a higher right ventricular systolic pressure (RVSP) (+ 69% at 15-minute peak, and 21-34% at 30-180 minutes), as well as a reduced mean arterial blood pressure (10-20% at 60-180 minutes), heart rate (20-26% at 60-180 minutes) and PaO 2 (9-11% at 30-180 minutes) compared with control rats. The role of the endothelin (ET) system, known to be involved in pulmonary hypertension of various etiologies, was investigated by evaluating the effect of the four classes of ET blockers: ET-converting enzyme inhibitor (ECEi) (CGS 35066), selective endothelin-A receptor antagonist (ET A -Ra) (Atrasentan, ABT-627), endothelin-B receptor antagonist (ET B -Ra) (A-192621) or mixed endothelin-A/endothelin-B receptor antagonist (ET A / B -Ra) (A-182086) in this animal model. All four were effective, to various degrees, at reducing the APAE-induced rise in RVSP. The relative efficacy of those compounds in reducing the acute elevation (15 minutes) of RVSP was ECEi ≥ ET A / B -Ra >> ET A -Ra = ET B -Ra. The sustained elevation (30-180 minutes) of RVSP was totally abolished by ECEi and attenuated by other ET blockers with a relative efficacy of ET A -Ra > ET A / B -Ra > ET B -Ra. ET receptor antagonists did not affect right ventricular basal tone (control rats) whereas ECEi reduced it by up to 12% after 2 hours. The APAE reduction in mean arterial blood pressure was unaffected by ETA-Ra, was completely normalized by ET B -Ra, but was further reduced by either ET A / B -Ra or ECEi. The basal mean arterial blood pressure in control rats was unaffected by ET A -Ra, was elevated by ET B -Ra, but was depressed by ET A / B -Ra and ECEi. All ET blockers maintained normal oxygen saturation in APAE. These results support a role for ETs in rat APAE, since ET blockers can attenuate the cardiopulmonary deterioration and blood gas exchange. However, modulation of the central hemodynamic profile is more complex and may limit the usefulness of some ET blockers.

Acute pulmonary air embolism (APAE) injures the vascular endothelium in the lung and results in pulmonary hypertension (PH). Endothelins (ETs), a family of potent vasoactive peptides, are known to be associated with PH of various aetiologies. We evaluated the effects of ABT-627, a selective ETA receptor (ETA-R) antagonist in a rat model of APAE over 3h. APAE rats developed a higher right ventricular systolic pressure (RVSP), lower mean arterial blood pressure (MABP), and had lower PaO2. At 3h, arterial plasma levels of ET-1 were increased. ABT-627-treated controls showed no effects. However, ABT-627 significantly lowered RVSP during APAE, abolished the short recovery phase (within 10–25min) of MABP without affecting the subsequent lowering of MABP, and improved oxygen saturation in APAE rats. These results show that ETA-R subtype is involved in the pathogenesis of APAE since a blockade of this receptor subtype attenuated the cardiopulmonary deterioration and improved blood gas exchanges in rats with this disease.

Postnatal mortality in isolated congenital diaphragmatic hernia (CDH) is mainly related to the associated pulmonary hypertension (PH) and to right-to-left shunting. Endothelins (ETs) are potent vasoconstrictors and pro-mitogenic peptides. Strong evidences support their participation in CDH and in the etiology of PH via the activation of ETA receptors (ETA-Rs). Evaluation of the effect of ABT-627, a selective non-peptidic ETA-R antagonist, given from −15 to 210 min post-delivery (1 mg kg−1 bolus +0.01 mg kg−1 h−1 infusion, i.v.), was conducted in the lamb model of CDH. Severity of CDH was assessed in comparison to untreated controls (n=5). Untreated CDH lambs (n=7) had a higher mean pulmonary arterial pressure (MPAP; P

Background and Purpose —Endothelins (ETs) are potent vasoconstrictors and may play a role in the pathophysiology of several diseases. Limited and controversial data exist on their role in human ischemic stroke. We planned a prospective, observational, and longitudinal clinical study to test whether ET-1 levels increase in various phases of ischemic stroke and whether the ET-1 levels correlate with neurological scores, stroke etiology, stroke risk factors, or final outcome. Methods —We measured plasma ET-1 levels with a sandwich-enzyme immunoassay method in 101 consecutive patients with ischemic stroke on admission and 1 week, 1 month, and 3 months after stroke and in 101 sex- and age-matched control subjects. At each sampling, the patients underwent a complete neurological evaluation. All stroke risk factors were recorded, an array of laboratory tests were performed, and the subtype of ischemic stroke was determined. The patients were contacted 3 years later for prognostic determination. Results —ET-1 levels in patients (2.4±1.3 pg/mL on admission, 2.2±1.4 pg/mL at 1 week, 2.1±1.4 pg/mL at 1 month, and 2.1±1.2 pg/mL at 3 months) were not different from those of the control subjects (2.2±0.9 pg/mL) at any time point. No correlation was found between the ET-1 levels and stroke etiology, stroke risk factors, stroke recurrence risk, age, sex, or neurological scores, except that ET-1 levels correlated with the use of warfarin and with body mass index. Conclusions —Plasma ET-1 levels were normal in patients with ischemic stroke. Our findings cannot exclude a role of ETs in the pathophysiology of ischemic stroke because plasma levels might not accurately reflect intracerebral concentrations, but they also do not support the occurrence of a major plasma ET-1 level increase at any phase of stroke. Our patient population is the largest ever reported in whom ET-1 levels were measured, but it consisted of mild and moderately ill patients with stroke due to the study design, of which the aim was long-term observation, which excludes severely ill patients.

Endothelin (ET) receptor antagonists may be beneficial for treating several medical conditions. Human trials with various ET receptor antagonists show that these antagonists elevate the plasma immunoreactive endothelin-1 (irET-1) level, and different classes of antagonists seem to affect the plasma ET-1 level differently. In this report, we study effects of ETA-selective, ETB-selective, and nonselective receptor antagonists on the plasma irET-1 level in the rat, and also compare available clinical data. The plasma irET-1 level was increased by five- and ten-fold after rats were treated with A-192621, an ETB-selective antagonist with Ki values for ETA and ETB at 5600 and 8.8 nM, for 3 days at 30 and 100 mg/kg/day via food. The plasma irET-1 level was increased by 1.8 and 2.4-fold when rats were treated with A-216546, an antagonist with Ki values for ETA and ETB at 0.46 and 13 000 nM, at 10 and 50 mg/kg/day via food for 7 days. As a comparison, the plasma irET-1 level was increased by > 24-fold when rats were treated with A-182086, a nonselective antagonist with Ki values for ETA and ETB at 0.2 and 1.2 nM, at 100 mg/kg/day via food for 9 days. In humans, blockade of ETA by ABT-627 did not result in an elevation in irET-1 until after 7 days of treatment. The results are consistent with the hypothesis that the ETB-receptor is the clearance receptor for ET-1. Our data also suggest that the modest effect of ETA antagonists on the plasma irET-1 level is probably a result of the upregulation of the ET-1 gene via a feedback mechanism.

An assay using scintillation proximity bead technology has been developed suitable for the quantitation of endothelin (ET) receptor antagonists in preclinical and clinical samples of plasma. The assay measures the competitive inhibition of radiolabelled ET-1 binding to ET(A) receptor membranes bound to wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads in the presence of plasma containing A-127722, a potent orally active, ET(A) selective ET antagonist. The assay requires as little as 50 microl plasma and no extraction procedure is needed. The SPA methodology eliminates the need for the separation of bound from free ligand. Using this method, A-127722 could be directly quantified in rat plasma with a detection limit of 1 ng/ml.

Tactile allodynia, the enhanced perception of pain in response to normally non-painful stimulation, represents a common complication of diabetic neuropathy. The activation of endothelin ET(A) receptors has been implicated in diabetes-induced reductions in peripheral neurovascularization and concomitant endoneurial hypoxia. Endothelin receptor activation has also been shown to alter the peripheral and central processing of nociceptive information. The present study was conducted to evaluate the antinociceptive effects of the novel endothelin ET(A) receptor-selective antagonist, 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N, N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627), in the streptozotocin-induced diabetic rat model of neuropathic pain. Rats were injected with 75 mg/kg streptozotocin (i. p.), and drug effects were assessed 8-12 weeks following streptozotocin treatment to allow for stabilization of blood glucose levels (>/=240 mg/dl) and tactile allodynia thresholds (

Endothelin-1 (ET-1) is thought to play a role in a wide range of pathological conditions. One of the distinct characteristics of ET-1 is its long-lasting vasoconstrictor action, which is presumably caused by the irreversible binding of ET-1 to ET receptors and by the functional effects of internalized ET receptors. ABT-627 is a potent endothelin-A (ET(A))-selective antagonist with a Ki value at 0.034 nM for the human ET(A) receptor, and is currently being used in clinical studies for prostate cancer. Unlike ET-1, the binding of 125I-labeled ABT-627 to human ET(A) receptors expressed in Chinese hamster ovary (CHO) cells is reversible, and the dissociation half-life for the ligand/receptor complex is 2 h. Interestingly, the binding of both ET-1 and ABT-627 to the ET(A)-receptor results in partial receptor internalization but only ET-1 is capable of triggering intracellular functional responses. Although ABT-627 binding to membranes is more reversible than ET-1 binding, ABT-627 is able to reverse an ET-1-induced contraction in rat aortic rings in a dose-dependent manner, and at 1 microM produces nearly complete reversal of the constrictor effects of 10 nM ET-1 within 60 min. Similarly, in vivo studies show that ABT-627 (0.01 and 0.1 mg/kg/min i.v.) reverses the ET-1-induced increase in arterial pressure in anesthetized, ganglionic-blocked rats, and after 60 min, ABT-627 essentially normalizes pressure. Our data show that ABT-627 is capable of reversing an established response induced by ET-1 and is useful in reversing pathological conditions involving ET-1.

We evaluated the role of endothelin-B- (ET(B)) receptor-mediated action in the development and maintenance of deoxycorticosterone acetate (DOCA)-salt-induced hypertension, cardiovascular hypertrophy and renal damage, using the spotting lethal (sl) rat which carries a naturally occurring deletion in the ET(B)-receptor gene. Homozygous (sl/sl) rats exhibit abnormal development of the neural crest-derived epidermal melanocytes and the enteric nervous system (ENS), and do not live beyond 1 month because of intestinal aganglionosis and resulting intestinal obstruction. Therefore, the dopamine-beta-hydroxylase (D betaH) promoter was used to direct ET(B) transgene expression in sl/sl rats to support normal ENS development. D betaH-ET(B) sl/sl rats live into adulthood and are healthy, expressing ET(B)-receptor in adrenals and other adrenergic neurons. When homozygous (sl/sl) and wild-type (WT) (+/+) rats, all of which were transgenic, were treated with DOCA and salt for 4 weeks, the homozygous rats exhibited significantly earlier and higher increases in systolic blood pressure than WT rats. The daily oral administration of ABT-627, a selective ET(A)-receptor antagonist, almost completely suppressed the DOCA-salt-induced hypertension in both groups. Renal dysfunction and histological damage induced by DOCA-salt treatment were more severe in homozygous than in WT rats. Increased and marked vascular hypertrophy of the aorta was also observed in homozygous rats, compared with WT rats. Renal and vascular injuries induced by DOCA and salt were significantly improved by ABT-627 administration. We propose that ET(B)-receptor-mediated actions are protective factors in the pathogenesis of DOCA-salt-induced hypertension. ET(A)-mediated actions are at least partly responsible for the increased susceptibility to DOCA-salt-induced hypertension and related tissue injuries in ET(B)-receptor-deficient rats.

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET A and ET B receptors. The ET A receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET B receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET A -selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET A -selective, ET B -selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET A selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.

Plasma concentrations of the vasoconstrictor endothelin-1 (ET-1) increase during acute physiologic stress, but the role of ET-1 in the pathophysiology of stress remains largely undefined. Whether ET-1 mediates thermoregulatory changes in vasomotor tone is unknown. ET-1 and its more stable precursor, Big ET-1, were measured in plasma obtained at several perioperative time points from 95 consecutive elderly patients (mean age 70 6 1 yr) randomized to receive either normothermic or hypothermic perioperative care while undergoing major surgical procedures. In the postoperative period, there were no significant changes in plasma ET-1 concentrations, but Big ET-1 concentrations increased considerably (P , 0.0001). There were no significant differences in mean ET-1 or Big ET-1 levels in normothermic and hypothermic patients. Preoperative and postoperative ET-1 concentrations were significantly higher in patients with a history of hypertension (P , 0.002) and in those requiring treatment for postoperative hypertension (P , 0.003). Patients with cancer and those undergoing abdominal surgery had significantly higher Big ET-1 concentrations (P , 0.0001 and P , 0.003, respectively). These data support the hypothesis that Big ET-1 is a more sensitive measure of endothelin system activation after major surgery. Premorbid conditions and location and type of surgery influence perioperative ET-1/ Big ET-1 concentrations. Implications: The endothelin response seems to be significantly associated with perioperative hemodynamic aberrations. The endothelin-1 (ET-1) precursor Big ET-1 is a more sensitive measure of the endothelin system activation in response to surgical stress than ET-1 alone. Thermoregulatory vasoconstriction in response to mild perioperative hypothermia occurs independently of the endothelin system. (Anesth Analg 1999;88:898‐903)

As a pharmacological class, Endothelin-A receptor (ET A ) antagonists are highly bound (> 98%) to serum albumin. In the presence of physiological concentrations of albumin, their affinities for ET A decrease 10 to 100 fold. We have prepared ET A antagonists which exhibit lower degrees of binding to albumin, while maintaining potency and selectivity for the ET A receptor. The protein induced IC 50 shift is reduced or eliminated in this new series of compounds. The compounds also display altered in vivo and pharmacokinetic profiles which may be consistent with their lower degree of protein binding.

We have previously shown that the endothelin (ET) isoforms ET- I and ET-3 and ET receptor antagonists exhibit a high degree (>98%) of binding to plasma proteins, especially serum albumin. This study examines the effects of human plasma proteins on ET- I binding and ET- I stimulated biologic responses. When ET- I binding to rat pituitary MMQ cells was examined, human serum albumin (HSA) inhibited ET-I binding in a concentration-dependent manner, with 92% inhibition observed at 5% (w/v) HSA. A similar observation was made when human plasma was tested. Surprisingly, addition of human plasma (0-100%) or HSA (0-5%) did not have a significant effect on ET-1-stimulated phosphatidylinositol (PI) hydrolysis in these cells. In human pericardial smooth-muscle cells. HSA again exhibited profound inhibitory effects on ET- I binding; 5% HSA inhibited ET-1 binding by 84%. However, ET- I-stimulated arachidonic acid release in these cells was not significantly affected by HSA (0-5%). Addition of increasing concentrations of human plasma incrementally decreased the potency of PD-156707, an ET A -selective antagonist, on inhibiting ET-1-stimulated PI hydrolysis in the MMQ cells. These results suggest that, although ET-I binding to the receptor is inhibited by human plasma proteins, especially HSA, ET-1-induced signal transduction is not significantly affected. Furthermore, plasma proteins decrease the potency of an ET receptor antagonist in inhibiting ET-1-stimulated biologic responses. This study may help to explain the discrepancy between in vitro and in vivo potencies of ET receptor antagonists.

Balloon angioplasty has become an important intervention in clinical cardiology; however, the technique is associated with a high incidence of restenosis, requiring repeated procedures. Endothelin-1 (ET-1), specifically through its action on ET(A) receptors, has been implicated in the cell proliferation and subsequent neointimal formation that leads to restenosis. Therefore we examined a potent antagonist of the ET(A) receptor, A127722.5, in a pig model of balloon angioplasty in iliac and carotid arteries. Ten pigs received A-127722.5 (7.5 mg/kg b.i.d.) orally, starting 3 days before angioplasty and continuing for 4 weeks; 10 additional pigs were treated with the same dosing regimen of the angiotensin-converting enzyme (ACE) inhibitor captopril (3.0 mg/kg b.i.d.), while a third group of 10 animals received placebo. At 2 and 4 weeks after the start of treatment, these doses of the ET(A) receptor antagonist and ACE inhibitor blocked the presser responses induced by big ET-1 and angiotensin I, respectively. In the iliac arteries, neointimal formation, neointimal/medial ratio, and maximal neointimal thickness were all significantly reduced, and the residual lumen area was significantly increased in pigs treated with the ET(A) receptor antagonist compared with placebo and captopril-treated groups. Medial collagen content, collagen deposition, and medial growth also were significantly reduced relative to the placebo group. Beneficial effects also were observed in the carotid arteries, although the results were less striking. Captopril was ineffective in protecting against the effects of balloon angioplasty in both vessels. Our results indicate that an orally active and potent antagonist of the ET(A) receptor inhibits cell proliferation and synthesis of extracellular matrix in pigs and may provide an important therapeutic approach to the prevention of restenosis.

To characterize the functional effect of endothelin-1 (ET1) and endothelin-3 (ET3), immunohistochemically localize ET1-like immunoreactivity, and measure the tissue levels of immunoreactive endothelin (irET) in canine genitourinary (GU) tissues.Canine GU tissues were characterized by measuring ET1 levels using a RIA, immunohistochemical staining of ET1 and isometric tension studies.Immunoreactive endothelin was present, to varying degrees, in the vas deferens, ureter, prostate, bladder and urethra. Functionally, ET1 demonstrated the typical concentration response characteristics in the canine bladder base, bladder body, and prostate. The maximal tension (Emax) measured following ET1 challenge was approximately 20-fold greater in the bladder body (0.67 +/- 0.21 g/mm.2) and bladder base (0.48 +/- 0.18 g/mm.2) as compared to the prostate 0.04 +/- 0.001 g/mm.2 The Emax of ET3 in the bladder body (0.31 +/- 0.12 g/mm.2) and bladder base (0.19 +/- 0.08 g/mm.2) was significantly lower than the corresponding Emax of ET1. No measurable contractile response was elicited by ET3 in the canine prostate. Immunohistochemical staining localized the ET-like immunoreactivity to the glandular epithelium of the prostate and the transitional epithelium of the bladder.Endothelins are ubiquitous in the canine lower GU tract with predominant localization to the epithelial elements. Endothelins are also functionally active in canine GU tissues, but the specific role of endothelins in the physiology and pathophysiology of GU tissues requires further investigation.

The human type-B endothelin receptor (h-ETB) was cloned from human lung poly A+RNA and stably expressed in CHO cells. Endothelin (ET) receptor binding and stimulation of PI hydrolysis demonstrated that the cloned h-ETB receptor is functional and linked to intracellular signal transduction pathways in CHO cells. The molecular mass of the h-ETB receptor was determined to be 65 KDa, and Bmax and Kd were 0.36 pmol/mg and 80 pM, respectively. Competition studies employing receptor ligands revealed that the potencies of the test ligands (IRL1620, PD142893, and Ro46-2005) were dependent on the length of the incubation time, whereas the natural agonists (ET-1 and ET-3) were not. When competing with ET-1 in the h-ETB receptor binding, the IC50 increased from 1.2 nM to 8.2 nM for IRL1620, 0.068 microM to 1.9 microM for PD142893, and 0.76 microM to 12.7 microM for Ro46-2005, as the incubation time increased from 1 hr to 24 hr. These time-induced changes are likely due to differences in the dissociation characteristics between the artificial ligands and the natural ligands.

The benzodioxole ((methylenedioxy)benzene) group is present in a number of endothelin (ET) receptor antagonists thus far reported. As part of our own endothelin antagonist program we have developed (2R*,3R*,4S*)-1-(N,N-dibutylacetamido)-4-(1,3-benzodioxol-5- yl)-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (A-127722). This is a potent antagonist, binding to the ETA and ETB receptor subtypes with affinities (IC50) of 0.4 and 520 nM, respectively, and also contains the aforementioned benzodioxole. While this compound was seemingly optimized at its N-terminus, no effort had been directed toward understanding the contributions to binding affinity or receptor subtype selectivity conferred by the benzodioxole. Substitution by 1- or 2-naphthyl yielded weak antagonists. Oxygenated benzenes, such as p-anisyl, were potent compounds with IC50s in the low-nanomolar range. Simple deletion of either of the two oxygen atoms (dihydrobenzofurans) yielded extremely potent agents, possessing subnanomolar affinity for the ETA receptor. Additionally, the compounds showed enhanced selectivity, binding to the ETB receptor subtype in the micromolar range. This paper describes the development of this novel class of compounds.

The N-terminal region of skeletal myosin light chain-1 (MLC-1) binds to the C terminus of actin, yet the functional significance of this interaction is unclear. We studied a fragment (MLC-pep; residues 5-14) of the ventricular MLC-1. When added to rat cardiac myofibrils, 10 nM MLC-pep induced a supramaximal increase in the MgATPase activity at submaximal Ca2+ levels with no effect at low and maximal Ca2+ levels. A nonsense, scrambled sequence peptide had no effect at any pCa value. MLC-pep did not affect myosin KEDTA and CaATPase activities or actin-activated MgATPase activities in the absence or presence of tropomyosin. The MLC-pep did not alter the ability of troponin I to inhibit MgATPase activity. Moreover, when troponin I and troponin C were extracted from the myofibrils, the MLC-pep lost its ability to stimulate the ATPase rate. This effect was fully restored upon reconstitution of the extracted myofibrils with troponin I-troponin C complex. Thus, activation of MgATPase activity by the peptide required a full complement of thin filament regulatory proteins. Interestingly, the stimulatory effect occurred at a ratio of 4 peptides to 1 thin filament, suggesting that the peptide engages in a highly cooperative process that may involve activation of the entire thin filament.

The effect of ETA receptor blockade on the bronchopulmonary response to endothelin-1 was determined in the airway of the anesthetized, spontaneously breathing guinea pig. Endothelin-1 administered as an aerosol increased lung resistance and decreased dynamic lung compliance. Delivery of the ETA receptor antagonist, FR139317, 5 min prior to giving endothelin-1 greatly potentiated these changes. A lower dose of endothelin-1 that had no effect on resistance or compliance produced large and significant changes when pretreated with FR139317. In contrast, aerosolized FR139317 had no effect on the bronchopulmonary response to intravenously administered endothelin-1. These data suggest a non-contractile function of ETA receptors accessible from the airways that serve to buffer the constrictor effects of non-ETA receptors.

The endothelin receptor subtypes involved in the vasoconstriction, capsular smooth muscle contraction, prostaglandin E2 and prostacyclin release induced by endothelin-1 have been investigated in the canine isolated perfused spleen using both the endothelin ETA receptor antagonist FR 139317 and the endothelin ETB receptor agonist IRL 1620. The isolated canine spleen was perfused with warmed (37°C) and oxygenated (95% O2/5% CO2) Krebs solution at constant flow with continuous recording of splenic arterial perfusion pressure and spleen weight. Samples of splenic venous effluent were collected to determine the amounts of prostaglandin E2 and prostacyclin, measured by radioimmunoassay. Endothelin-1 (4–200 pmol) and IRL 1620 (20–1000 pmol) dose-dependently increased splenic arterial perfusion pressure but the former was more potent on a molar basis (the molar dose ratio IRL/endothelin-1 required to increase splenic arterial vascular resistance by 25% was approximately 33). The infusion of the nitric oxide inibitor Nω-nitro- l -arginine methyl ester (10 μM), but not of the enantiomer Nω-nitro- l -arginine methyl ester, significantly potentiated the increase in splenic arterial vascular resistance induced by endothelin-1. The infusion of FR 139317 (1 μM) markedly attenuated the increased splenic arterial perfusion pressure induced by endothelin-1 without affecting that evoked by IRL 1620. At the highest dose (200 pmol), endothelin-1 induced a small but significant capsule contraction as reflected by the reduction in the spleen weight. The infusion of FR 139317 (1 μM) abolished this contractile effect. IRL 1620 (in doses up to 1000 pmol) did not significantly affect the capsule tone. The administration of either endothelin-1 (20–200 pmol) or IRL 1620 (20–1000 pmol) caused the release of 6-oxo-prostaglandin F1α (breakdown product of prostacyclin) and prostaglandin E2 into the splenic venous effluent. The amount of both prostanoids released by endothelin-1 was significantly greater than that induced by IRL 1620. FR 139317 (1 μM) significantly reduced (P

Endothelin (ET) binding to receptors has been shown to be almost irreversible. We studied the dissociation characteristics of ET A receptor agonists and antagonists using membranes prepared from rat pituitary cells (MMQ). Consistent with our previous report, competition studies comparing ET-1, ET-3, BQ123, FR139317, PD142893, and Ro46-2005 show that MMQ cells contained predominantly the ET A receptor. [ 125 I]ET-1 bound to the receptor was difficult to dissociate. In contrast, bound BQ123, FR139317, and Ro46-2005 were easier to dissociate, suggesting that antagonist binding was more reversible. Although BQ123 (5 nM), FR139317 (1 nM), and Ro46-2005 (0.5 μM) inhibited 0.1 nM [ 125 I]ET-1 binding by greater than 80% after 15 min of incubation, the inhibition decreased to less than 20% after 24 h of incubation. This decrease in binding inhibition was not the result of antagonist degradation. These results suggest that, similar to our previous observation made with the ET B receptor in porcine cerebellum, the ability of antagonists to inhibit [ 125 I]ET-1 binding to the ET A receptor is critically dependent on the incubation time because of the difference in the dissociation characteristics between antagonists and ET-I.

A series of 5-membered ring heterocyclic analogs of A-81988 were synthesized in order to determine their activity as angiotensin II antagonists. The activity of these compounds in a rabbit aorta in vitro assay ranged from pA2 values of

Fused ring heterocyclic analogs of A-81080 (pA2= 9.9) were synthesized and their activities in the rabbit aorta in vitro assay were measued. The best compounds (pA2 = 8.6) in series 1 had R1 = Et, R2 = H, W = X = Z = C-H, and Y = C-OMe or C-COOH. In series 2, the best compound (pA2 = 8.3) had R1 = Et, R2 = H, W = N-Me, X = C-H, and Y = N.

Endothelins mediate contractile responses in many types of vascular and nonvascular smooth muscle. The present study represents the first detailed characterization of endothelins in the human prostate. The objectives of this study were to determine the tissue levels and source of endogenous endothelin-1 (ET1) in the human prostate. The contractile effects of ET1 were also investigated using in vitro isometric tension studies. The mean tissue level of ET1 was 0.58 +/- 0.08 pg./mg. tissue wet weight. Endothelin-like activity was markedly prominent in the glandular epithelium of the human prostate, whereas minimal endothelin-like activity was observed in the prostatic stroma. Strips of human prostatic tissue were suspended in isolated tissue chambers and challenged to a concentration response of ET1. The mean EC50 and Emax for ET1 was 3.2 x 10(-8) M. and 0.12 +/- 0.02 gm. force per mm.2 cross-sectional area (CSA), respectively. Preincubation with indomethacin, terazosin, or nifedipine did not alter the concentration-dependent response to ET1. A calcium-free buffer abolished the contractile response to ET1. Thus, ET1 mediates a potent contraction of human prostatic smooth muscle that is not mediated via alpha 1 adrenergic or dihydropyridine sensitive calcium channels or prostaglandin synthesis. The presence of marked endothelin-like immunoreactivity strongly suggests a biological significance for endogenous endothelins in the human prostate.

Blockade of the renin-angiotensin system was studied in male Sprague-Dawley rats during long-term inhibition of nitric oxide synthase. Nitro-L-arginine-methyl ester (L-NAME) was placed in the drinking water for 4 weeks (approximately 100 mg/kg per day). Separate groups of rats were coadministered the angiotensin II antagonist A-81988 in the drinking water ranging from approximately 0.001 to 1 mg/kg per day. Control groups received only tap water or A-81988 alone. Each week, rats were placed in metabolic cages, and tail-cuff blood pressures and blood samples were taken. L-NAME produced a sustained elevation in tail-cuff pressure that was completely prevented by A-81988. No changes in creatinine clearance, sodium excretion, plasma creatinine concentration, or blood urea nitrogen were observed. Food and water intakes were identical in all groups. Water excretion was significantly increased in L-NAME-treated animals regardless of additional inhibitor treatment, suggesting a possible role for nitric oxide synthase in the control of water excretion; this effect was independent of blood pressure. Although less potent than A-81988, the angiotensin II antagonist losartan and the angiotensin converting enzyme inhibitor enalapril also blocked L-NAME-induced hypertension. In a separate series of experiments, rats were not given A-81988 until 2 weeks after hypertension had fully developed in L-NAME-treated rats. Within 1 week of treatment with the angiotensin II antagonist, tail-cuff pressure returned to normal. We conclude from these studies that long-term inhibition of endogenous nitric oxide production produces an angiotensin II-dependent form of hypertension.

Experiments were conducted to characterize the nature of a phosphoramidon-sensitive endothelin-converting enzyme in vivo by evaluating the pressor response to a bolus intravenous (i.v.) injection of endothelin family peptides following administration of phosphoramidon in anesthetized Sprague-Dawley rats. Phosphoramidon given i.v. at 10 mg/kg completely prevented the pressor response to human big endothelin-1-(1-38) (big ET-1). The EC50 for phosphoramidon was determined to be in the range of 1 to 3 mg/kg. The pressor response to big ET-1 60 min after phosphoramidon injection was attenuated by roughly 60% indicating a long inhibitory half-life. Very high doses of big ET-1 (> 20 mg/kg) were capable of over-riding the effect of phosphoramidon and produced characteristic pressor responses suggesting that the inhibition by phosphoramidon can be considered competitive in nature. Human big endothelin-3-(1-41) (big ET-3) produced significant increases in arterial pressure although with less potency and efficacy compared to big ET-1. The pressor response to big ET-3 was also inhibited by phosphoramidon. Phosphoramidon does not act indirectly by interfering with ET-1 receptor-mediated actions since the inhibitor has no effect on the in vivo pressor response to ET-1 and does not antagonize [125I]ET-1 receptor binding or constrictor responses in vitro. These results are consistent with the idea that a phosphoramidon-sensitive endothelin-converting enzyme is capable of cleaving both big ET-1 and big ET-3 to the active peptides in the rat.

Experiments were conducted to determine the ability of the newly discovered ETA receptor antagonist, BQ-123, to inhibit the pressor responses to several members of the human endothelin peptide family in anesthetized rats. Big ET-1 produced a greater increase in mean arterial pressure compared to ET-1 (67 ± 10% vs. 44 ± 4%, p < 0.05; both at 1 nmol/kg i.v.). Prior administration of BQ-123 (10 mg/kg i.v.) was more effective at inhibiting the response to Big ET-1 compared to ET-1. Following ETA receptor blockade, ET-1 produced a small, but significant, increase in mean arterial pressure (15 ± 2%) while Big ET-1 had no effect (5 ± 2%). Big ET-3 and ET-3 at 1 nmol/kg i.v. produced similar increases in mean arterial pressure, although less than that produced by either Big ET-1 or ET-1 (p < 0.05). ETA receptor antagonism blocked 64% of the pressor response to both Big ET-3 and ET-3. We conclude that blockade of pressor responses to each of the endothelin peptides suggests that the increases in arterial pressure ...

Experiments were designed to examine the role of endothelin (ET) receptors, specifically ETA receptors, in mediating the renal vasoconstrictor effects of ET-1 in anesthetized Sprague-Dawley rats. Intravenous infusion of ET-1 at 25 pmol.kg-1 x min-1 for 60 min produced a significant increase in mean arterial pressure (20 +/- 7%) and decreases in renal plasma flow (-60 +/- 6%) and glomerular filtration rate (-47 +/- 6%). Renal vascular resistance was significantly increased from 17 +/- 1 mmHg.ml-1 x min.g kidney wt during control period to 54 +/- 11 mmHg.ml-1 x min.g kidney wt during the experimental period. A second group of rats was infused with both ET-1 and the specific ETA receptor antagonist BQ-123 (0.1 mg.kg-1 x min-1). ET-1-induced increases in mean arterial pressure were completely blocked by BQ-123 (the average change was -7 +/- 4%). However, the renal vasoconstrictor effects of ET-1 were not affected by the antagonist, since renal plasma flow and glomerular filtration rate were again significantly reduced (-54 +/- 4 and -56 +/- 6%, respectively). Once again, renal vascular resistance was significantly increased from 16 +/- 2 mmHg.ml-1 x min.g kidney wt during the control period to 33 +/- 5 mmHg.ml-1 x min.g kidney wt during the experimental period. In a third group, infusion of BQ-123 alone produced a significant decline in mean arterial pressure (-13 +/- 2%), with no significant changes in renal plasma flow or glomerular filtration rate, thus producing a significant decrease in renal vascular resistance (15 +/- 1 vs. 11 +/- 2 mmHg.ml-1 x min.g kidney wt).(ABSTRACT TRUNCATED AT 250 WORDS)

The importance of glycosylation to endothelin (ET) receptors was studied in cultured Swiss 3T3 fibroblasts. ET stimulated phosphatidylinositol hydrolysis by 270% in control cells. This stimulatory effect was decreased to < 130% in tunicamycin (TU)-treated cells. TU inhibited protein glycosylation, but did not effect DNA synthesis, cell morphology, or cell numbers. ET-1 binding was greatly reduced in TU-treated cells. In membranes prepared from control cells, [125]ET-1 binding was saturable, and appeared as a straight line in Scatchard analysis with values of 0.7 pmol/mg and 0.9 nM for Bmax and KD, respectively. [125]ET-1 binding to membranes from TU-treated cells was not saturable. Scatchard analysis showed a biphasic line. Values of Bmax, and KD for the higher affinity site were 0.17 pmol/mg and 0.12 nM, respectively. The low affinity site exhibited a much weaker affinity for ET with an estimated KD value of 6.8 nM. Cross-linking studies revealed that the receptor in control membranes was a protein with ...

Although many agents have been shown to stimulate release of endothelin (ET) in vitro, the factors regulating and affecting production and release of ET in intact animals remain obscure. Experiments were conducted to determine the effect of surgical preparation and infusion of plasma and/or saline on circulating immunoreactive (ir)-ET concentrations and to characterize the mechanism of endotoxin-induced increases in plasma ir-ET. Male Sprague-Dawley rats (210-325 g) anesthetized with Inactin were placed on a surgical heating table for a period of about 2 h before blood was collected via puncture of the abdominal aorta. The plasma concentration of ir-ET was 8.1 +/- 0.9 pg/ml in rats that did not undergo surgical preparation or receive any further treatment. Standard surgical preparation, as for renal clearance experiments (catheters in the trachea, femoral artery, femoral vein, and bladder), resulted in a dramatic rise in ir-ET to 17.7 +/- 3.0 pg/ml (p0.05). Infusion of plasma from donor rats (5 ml/kg over 20 min) resulted in an additional significant increase to 32.6 +/- 3.5 pg/ml (p0.05). In contrast, infusion of saline (0.9% NaCl) in a similar manner produced no further increase in ir-ET levels (23.2 +/- 3.2 pg/ml). Infusion of endotoxin in anesthetized, surgically prepared rats significantly increased ir-ET. This increase was not blocked by a platelet-activating factor antagonist (Esai 6123), despite blockade of endotoxin-induced hypotension. Phosphoramidon, which has been shown to block in vivo conversion of exogenous big ET to ET, was unable to prevent endotoxin-induced increases in ir-ET.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments were conducted to determine the effect of surgical preparation and infusion of plasma and/or saline on circulating immunoreactive endothelin (irET) concentrations. Male Sprague-Dawley rats anesthetized with Inactin were placed on a surgical heating table for about 2 h before collecting blood via puncture of the abdominal aorta. The plasma concentration of irET was 8.1 ± 0.9 pg/ml in rats that did not undergo surgical preparation or receive any further treatment. Standard surgical preparation as for renal clearance experiments (catheters in the trachea, femoral artery, femoral vein, and bladder) resulted in a dramatic rise in irET to 17.7 ± 3.0 pg/ml (p < 0.05). Infusion of plasma (5 ml/kg over 20 min), but not saline, resulted in an additional increase to 32.6 • 3.5 pg/ml (p < 0.05). These results clearly demonstrate that standard whole-animal techniques such as surgery and plasma infusion produce significant changes in circulating endothelin which may affect a variety of physiological and pha...

Experiments were conducted to determine the importance of the carbohydrate moiety of phosphoramidon in the inhibition of the pressor response to big endothelin-1 in anesthetized rats. Big endothelin-1 produced a 42% increase in mean arterial pressure which was nearly abolished by co-infusion of phosphoramidon. Similarly, when an analog of phosphoramidon lacking the rhamnose group (phosphoryl-leu-trp-OH) was co-infused, a significant attenuation of the pressor response was observed. These findings indicate that the rhamnose moiety of phosphoramidon is not responsible for distinguishing this compound as an inhibitor of the response to big endothelin-1 in the rat.

The putative endothelin-converting enzyme (ECE) has been the focus of intense research, both within academia and the pharmaceutical industry. Interest in ECE stems mainly from the hypothesis that development of inhibitors of ECE will provide an effective means of preventing production of endothelin in circumstances where it may play a pathogenic role. Both an aspartic and a metalloprotease have been identified that have characteristics of this putative enzyme. Evidence suggests that the metalloprotease, which is inhibited by phosphoramidon, may be the physiologically relevant converting enzyme. However, it remains to be demonstrated conclusively that any inhibitor of an ECE activity directly alters endogenous endothelin production and/or the pathogenesis of a disease condition in which endothelin is thought to play a primary role.

We have recently reported that administration of the ANF analog A68828 improves renal function in an acute model of postischemic acute renal failure. The current investigation examined the question whether a short-term infusion of A68828 can attenuate the long-term decrement in renal function following an ischemic event. Acute renal failure was induced in male Sprague-Dawley rats (200-250 g) by complete occlusion of both renal arteries for 30 min. During the initial 60 min following ischemia, vehicle (0.1% BSA in saline), A68828 (10 micrograms/kg/min); dopamine (10 micrograms/kg/min); A68828 (10 micrograms/kg/min) plus dopamine (10 micrograms/kg/min); or ANF[1-28] (0.5 microgram/kg/min) were infused intravenously. In vehicle-treated animals, a very large increase in plasma creatinine was observed, with peak levels at 2 days postischemia (5.5 +/- 1.2 mg/dL). A68828 alone, A68828 with dopamine, or ANF[1-28] infusion attenuated the rise in plasma creatinine levels by approximately 50% on each day of the study; dopamine alone was no different from vehicle-treated controls. Gross histological examination of kidneys on the fourth day postischemia revealed that significantly less damage occurred only in the group treated with A68828 alone. These results indicate that infusion of a reduced-size analog of ANF for a brief period in the postischemic kidney improves renal function and lessens tissue damage as evaluated several days after the ischemic event. Furthermore, dopamine infusion provides no discernable beneficial effect.

We compared the hemodynamic effects of continuous i.v. infusion of endothelin-1 and big endothelin-1 in anesthetized rats. Big endothelin-1 was fivefold less potent than endothelin-1 in decreasing cardiac output, heart rate, and stroke volume. However, big endothelin-1 produced a significantly larger increase in mean arterial pressure compared to endothelin-1 at doses that produced identical decreases in cardiac output. These findings support the hypothesis that the hypertensive effects of big endothelin-1 and endothelin-1 are produced by differential effects on systemic vascular resistances.

Endothelin-1 (ET-1) was originally identified in the culture supernatant of porcine aortic endothelial cells. From the deduced amino acid sequence, a biosynthetic pathway has been proposed that a prepro- form of porcine ET-1 is initially processed by dibasic pair proteolysis to a 39-amino acid intermediate form (big ET), which is then converted to ET-1 by specific proteolytic cleavage between Trp21 and Val22. We have identified an enzyme activity that converts human big endothelin[1-38] to endothelin[1-21] and a C-terminal fragment (CTF, 22-38) in a homogenate fraction from rat lung. The conversion activity was enriched threefold in a plasma membrane fraction. Metal ions activated the activity by about 1.5- to 2.5-fold, in the order of Mn2+ greater than Zn2+ = Ca2+ greater than Mg2+ greater than Ba2+. The conversion activity was optimal at pH 4.0, was inhibited by pepstatin-A (IC50 = 20 nmol), but not affected by TLCK, aprotinin, PMSF, E-64, bestatin, phosphoramidon, or thiorphan at 40 microM. The converting enzyme was partially purified from rat lung plasma membranes by sequential HPLC on Mono Q, Superose 12, and Mono P. The enzyme has an apparent molecular mass of 90 kDa as estimated by SDS-PAGE or gel filtration and appears to be a single peptide protein. The enzyme may exist as isozymes with isoelectric point (pI) values at 6.2 and 6.3.

Endothelin-1 (ET-1) is reported to be the most powerful constrictor of blood vessels known. Atrial natriuretic factor is a potent relaxor of contracted vessels. This study examined the potential interaction between these vasoactive peptides on rabbit aortic rings. ET-1 contracted the rings in a dose-dependent manner with an ec50 (-log M) of 9.78 ± 0.16. Atriopeptin III (APIII) completely relaxed ET-1 (10−9 M)-contracted rings with an EC50 of 9.63 ± 0.07 and methoxamine contracted rings with an EC50 of 9.18 ± 0.09. Pretreatment of aortic rings with APIII (up to 3 × 10−6 M) did not alter the normal ET-1 dose-response curve with respect to potency but did diminish the maximal contraction achieved. An interesting additional finding was that the temporal nature of ET-1-induced contraction is remarkably dose-variable. In conclusion, the potent contractile effects of ET-1 on vascular smooth muscle can be effectively reversed but not prevented by APIII.

An enzyme activity which converts human big endothelin (1-38) to endothelin (1-21) and a C-terminal fragment (CTF, 22-38) was identified in a plasma membrane fraction prepared from rat lung. The conversion activity was optimal at pH 4.0, was inhibited by Pepstatin-A (IC50 = 20 nM), but was not affected by TLCK, Aprotinin, PMSF, E-64, Bestatin, Phosphoramidon or Thiorphan at 40 microM. Metal ions activated the activity by 1.5 - 2.5 fold in the order of Mn+2 greater than Zn+2 = Ca+2 greater than Ba+2. These data suggest that a Pepstatin-A inhibitable, metal ion related aspartic protease may be involved in the conversion of big endothelin to endothelin in rat lung.

This study evaluated the effects of rat ANP(5–28) infusion into the blood-perfused dog gracilis muscle at concentrations ranging from 30 to 10,000 pg/ml. The vasculature of gracilis muscles from anesthetized beagle dogs was isolated and pump-perfused at constant flow with blood utilizing an extracorporeal circuit. Maximal vasodilatory capacity was determined by adenosine injection. ANP was infused into the arterial circuit to produce increasing arterial blood concentrations. Each infusion lasted 10 min. Systemic arterial pressure, central venous pressure, cardiac output and heart rate did not change during ANP infusion into the gracilis vasculature. ANP at arterial blood concentrations up to 10,000 pg/ml did not produce significant vasodilation although the vasculature showed pronounced vasodilation in response to adenosine. In vitro experiments showed that ANP had much less vasorelaxant activity in dog femoral artery and saphenous vein than in rabbit aorta. Therefore, rat ANP(5–28) at concentrations within and well above physiological and pharmacological ranges does not inhibit the basal vascular tone present in the innervated, blood-perfused dog gracilis muscle in situ .

The role of endothelin-B (ETB) 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 ETB blockade and the role of ETA 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 ETB-selective antagonist A-192621 (0.1, 1.0, and 10 mg/kg bid, 4 days/dose) followed by coadministration of the ETAantagonist atrasentan (5 mg/kg bid) + A-192621 (10 mg/kg bid) for another 4 days. High-dose ETB 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 ETB blockade produces a sustained hypertension in conscious nonhuman primates, which is mediated by ETA receptors. These data suggest an importance clearance function for ETB receptors, one that influences arterial pressure homeostasis indirectly by reducing plasma ET-1 levels and minimizing ETA activation.

Asthma is a chronic respiratory disease that is characterized by airway inflammation, bronchoconstriction and the influx of pro-inflammatory cells, mostly eosinophils in the lung tissue and bronchoalveolar space. Amongst the many physiopathological roles attributed to endothelins (ETs), one is to modulate pulmonary functions. It is established that Balb/c mice develop allergen-induced Th2-cytokine gene expression, airway inflammation and hyper-responsiveness whereas C57Bl/6 mice are much less reactive. In the present study, we investigated the roles of ETs in these two murine models of allergic asthma (AA). Mice were sensitized and challenged with either saline (S) and/or ovalbumin (O) over 6 weeks (groups S/S and O/O) and treated chronically with ABT-627 or its vehicle. Twenty-four hours after the last sensitization, challenged mice developed a marked airway inflammatory response characterized by the accumulation of total inflammatory cells (a 21-fold increase) in the bronchoalveolar space, similar in both mouse strains. The increase in eosinophils was marked in both groups, representing 98% of total cell count in O/O versus

Endothelins (ETs), 21-amino-acid peptides involved in the pathogenesis of various diseases, bind to ETA and ETB receptors to initiate their effects. Based on the same core structure, we have developed four small-molecule ET receptor antagonists, ABT-627 (atrasentan), ABT-546, A-182086 and A-192621, which exhibit differences in selectivity for ETA and ETB receptors. In this report, we compare the efficacy, potency and pharmacokinetic properties of these four antagonists, including potency in inhibiting ET-1- or Sarafotoxin 6c-induced vessel constriction in isolated arteries and efficacy in antagonizing ET-1-, big ET-1- or Sarafotoxin 6c-induced pressor responses in rats.

Endothelins (ETs), 21-amino-acid peptides involved in the pathogenesis of various diseases, bind to ETA and ETB receptors to initiate their effects. Based on the same core structure, we have developed four small-molecule ET receptor antagonists, ABT-627, ABT-546, A-182086 and A-192621, which exhibit difference in selectivity for ETA and ETB receptors. In this report, we compare the potency and selectivity of these four antagonists in inhibiting 125I-labelled ET-1 binding to cloned human ETA and ETB receptors, and in blocking ET-1-induced functional responses (arachidonic acid release and phosphatidylinositol hydrolysis).

The synthesis and structure-activity relationships (SAR) of a series of pyrrolidine-3-carboxylic acids as endothelin antagonists are described. The data shows an increase in selectivity when the methoxy of Atrasentan (ABT-627) is replaced with methyl, and the benzodioxole is replaced with dihydrobenzofuran. Adding a fluorine further increases the binding activity and provides a metabolically stable and orally bioavailable ET(A)-selective antagonist.

Background —Endothelin (ET)-1 plays an important role in the pathogenesis of deoxycorticosterone acetate (DOCA)-salt–induced hypertension. We evaluated the pathological role of ET B receptors in DOCA-salt–induced hypertension, cardiovascular hypertrophy, and renal damage by using the spotting-lethal ( sl ) rat, which carries a naturally occurring deletion in the ET B receptor gene. Methods and Results —Homozygous ( sl / sl ) rats exhibit abnormal development of neural crest–derived epidermal melanocytes and the enteric nervous system, and they do not live beyond 1 month because of intestinal aganglionosis and intestinal obstruction. The dopamine β-hydroxylase (DβH) promoter was used to direct ET B transgene expression in sl / sl rats to support normal enteric nervous system development. DβH-ET B sl/sl rats live into adulthood and are healthy, expressing ET B receptors in adrenal glands and other adrenergic neurons. When homozygous ( sl / sl ) and wild-type (+/+) rats, all of which were transgenic, were treated with DOCA-salt, homozygous rats exhibited earlier and higher increases in systolic blood pressure than did wild-type rats. Chronic treatment with ABT-627, an ET A receptor antagonist, completely suppressed DOCA-salt–induced hypertension in both groups. Renal dysfunction and histological damage were more severe in homozygous than in wild-type rats. Marked vascular hypertrophy was observed in homozygous rats than in wild-type rats. Renal and vascular injuries were significantly improved by ABT-627. In DOCA-salt–treated homozygous rats, there were notable increases in renal, urinary, and aortic ET-1, all of which were normalized by ABT-627. Conclusions —ET B -mediated actions are protective in the pathogenesis of DOCA-salt–induced hypertension. Enhanced ET-1 production and ET A -mediated actions are responsible for the increased susceptibility to DOCA-salt hypertension and tissue injuries in ET B receptor–deficient rats.

This study was designed to quantify the long-term contribution of endogenous endothelin-1 (ET-1) and ETAreceptors to the regulation of arterial pressure under normal conditions in nonhuman primates. Therefore, mean arterial pressure (MAP) and heart rate were measured 24 h/day with the use of telemetry techniques in conscious cynomolgus monkeys under control conditions, during administration of an ETAselective receptor antagonist (ABT-627; 5 mg/kg, 2 times a day by mouth, 4 days), and a 6-day posttreatment period. Systemic ETAblockade reduced MAP (24 h) from 89 ± 3 to 82 ± 2 and 79 ± 2 mmHg on days 1 and 4, respectively. Subsequently, MAP remained suppressed for 3 days posttreatment. Heart rate increased from 111 ± 5 to 122 ± 4 and 128 ± 6 beats/min on days 1and 4 of ABT-627, respectively, and remained above control for 3 days posttreatment. Plasma ET-1 concentration increased from 1.0 ± 0.3 to 1.9 ± 0.4 pg/ml in response to ABT-627 ( day 4) but decreased to control values 4 days posttreatment. These data demonstrate a physiologically important role for endogenous ET-1 and ETAreceptors in the long-term regulation of arterial pressure and plasma ET-1 levels in the conscious nonhuman primate.

We investigated the effects of ABT-627, a selective ETA-receptor antagonist, and A-192621, a selective ETB-receptor antagonist, on ischemic acute renal failure (ARF) in rats. Ischemic ARF was induced by clamping the left renal artery and vein for 45 min, 2 weeks after the contralateral nephrectomy. Renal function in untreated ARF rats markedly decreased at 24 h after reperfusion and thereafter tended to recover gradually. ABT-627 (1 mg/kg, i.v.) administration before ischemia markedly attenuated the renal dysfunction induced by the ischemia/reperfusion, whereas A-192621 (3 mg/kg, i.v.) pretreatment was without effect. Histopathological examination of the kidney of untreated ARF rats revealed severe renal damage such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion. Histologically evident damage was improved by pretreatment with ABT-627, but not with A-192621. Daily oral administratrion of ABT-627 (10 mg/kg per day), but not A-192621 (30 mg/kg per day), given after the ischemia/reperfusion period also exerted protective effects. These findings clearly indicate that endothelin, acting via the ETA receptor, participates in the pathogenesis of ischemic ARF. Thus, selective ETA-receptor antagonism may be useful in the treatment of human ischemic ARF, whereas selective blockade of the ETB receptor will probably be ineffective.