Subtype-specific desensitization of human endothelin ETA and ETB receptors reflects differential receptor phosphorylation

Endothelins regulate blood pressure in mammals through G protein-coupled receptors. Two receptor subtypes, ETA and ETB, exist which differ by their agonist profiles. Here we show subtype-specific differences in the inactivation of these endothelin receptors. Using a modified inositol phosphate accumulation assay, we found that stimulation of ETA by endothelin-1 results in sustained activation of the subtype, retaining >30% of its initial activity even 20 min after agonist administration, whereas the ETB rapidly deactivated after agonist stimulation, losing >80% of its initial activity within 5 min after endothelin application. The discrepancy in receptor inactivation is reflected by subtype-specific differences in receptor phosphorylation. Whereas ETA failed to undergo ligand-induced phosphorylation, the ETB was rapidly phosphorylated in response to agonist stimulation. By contrast, the kinetics of ligand-induced internalization were essentially identical for the receptor subtypes, suggesting endothelin receptor internalization being independent of ligand-induced receptor phosphorylation. Interestingly, a strong correlation was observed between the time course of ETA inactivation and ETA internalization. Therefore, our data suggest a subtype-specific inactivation of human endothelin receptors: fast receptor phosphorylation in the case of ETB and slow receptor internalization in the case of ETA. Subtype-specific modulation of endothelin receptors may account for the short-term hypotensive effects of endothelins via rapidly down-regulating ETB receptors and the long-lasting hypertensive effects due to sustained ETA activation.     

Comparison of the acute effects of a selective endothelin ETA and a mixed ETA/ETB receptor antagonist in heart failure

OBJECTIVE: Both the selective endothelin (ET) ETA receptor and mixed ETA/ETB receptor antagonists improve haemodynamics in patients and experimental models with congestive heart failure (CHF) and reduce the mortality in CHF rats. However, it remains unclear which of these antagonists is superior in the treatment of CHF. In addition, there is little information as to whether these ET receptor antagonists contribute to the neuroendocrine regulation and body fluid balance. We therefore investigated the cardiorenal and neurohumoral benefits of selective ETA receptor and mixed ETA/ETB receptor antagonists in CHF. METHODS: We administered acutely either the selective ETA receptor antagonist FR139317 (FR, n = 6, 1 and 10 mg/kg) or the mixed ETA/ETB receptor antagonist TAK-044 (TAK, n = 6, 1 and 3 mg/kg) to conscious dogs with CHF induced by rapid right ventricular pacing for ten days. RESULTS: Both FR and TAK decreased the cardiac pressures and the plasma atrial natriuretic peptide level and increased the cardiac output and urinary sodium excretion. FR increased the urine flow rate in association with an increased glomerular filtration rate and renal plasma flow, while TAK reduced the plasma aldosterone level. Neither antagonist increased the plasma renin activity or norepinephrine levels. CONCLUSIONS: These ETA/ETB antagonist. However, the long-term administration of a mixed ETA/ETB receptor antagonist would improve not only the haemodynamics but also prevent fluid retention by suppressing secretion of aldosterone during the treatment of chronic CHF.     

Expression of endothelin(A) receptors in human gliomas and meningiomas, with high affinity for the selective antagonist PD156707

OBJECTIVE: Endothelin (ET) immunoreactivity, ET production, and specific ET receptors have been identified in the brain. Changes in ET concentration or receptor expression have been implicated in the pathophysiological changes in vasospasm after subarachnoid hemorrhage and in cerebral neoplasia. In this study, we have characterized the ET(A) and ET(B) receptor subtypes present in human normal cerebral cortex (NCC) and two common central nervous system tumors, i.e., meningioma (MNG) and glioblastoma multiforme (GBM). A knowledge of the ET receptor subtypes present may provide a novel therapeutic target for newly developed ET antagonists. METHODS: Saturation, competition, and autoradiographic studies were performed with the subtype-specific radioligands 125I-PD151242 and 125I-BQ3020, to characterize the ET(A) and ET(B) receptors present in NCC, MNG, and GBM. RESULTS: NCC expresses high-affinity ET(A) receptors on pial and intraparenchymal vessels and high-affinity ET(B) receptors on glia and neurons. MNGs express mainly (85%) high-affinity ET(A) receptors in a diffuse pattern, whereas GBMs express high-affinity ET(A) receptors on the neovasculature and ET(B) receptors on the nonvascular elements. The ET antagonist PD156707 (Kd = 0.059 nmol/L) showed a higher affinity for the ET(A) receptor subtype than did bosentan (Kd = 1.1 nmol/L). CONCLUSION: ET(A) receptors are expressed in high concentrations in MNGs and in the vasculature of NCC and GBMs. The ET(A)-selective antagonist PD156707 may be of potential therapeutic value in vascular and neoplastic diseases of the central nervous system.     

Effects of nitric oxide synthase inhibition and endothelin ETA receptor blockade on haemodynamics in hypertensive rats

1. The objectives of the present study were to study regional differences in haemodynamics between spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats induced by the nitric oxide synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) and the endothelin ETA receptor antagonist BQ 123 in vivo in tissues known to be important for blood pressure (BP) regulation (heart, kidney and skeletal muscle). Furthermore, the effect of acetylcholine (ACh) infusion (2 micrograms/kg per min) was examined after L-NMMA or BQ 123. The microsphere method was used for determinations of cardiac index (CI) and regional haemodynamics. 2. NG-Monomethyl-L-arginine (20 mg/kg) increased BP (26-48%; P < 0.01) and reduced CI in both rat strains. BQ 123 (1 mg/kg) reduced BP slightly (-4 to 11%; P < 0.05). 3. NG-Monomethyl-L-arginine significantly increased myocardial and skeletal muscle vascular resistance in SHR only; however, in the kidney, L-NMMA reduced blood flow and increased vascular resistance in both rat strains. 4. BQ 123 induced minor changes in regional haemodynamics that were not significantly different between the two strains. 5. Acetylcholine following BQ 123 induced an increase in myocardial blood flow in WKY rats, but decreased blood flow in SHR. Acetylcholine following L-NMMA reduced myocardial blood flow in both strains. 6. Acetylcholine following BQ 123 induced renal vasodilation in WKY rats but, following L-NMMA, ACh did not induce renal vasodilation in either rat strain. In contrast, L-NMMA did not abolish the vasodilation of acetylcholine in skeletal muscle in WKY rats. 7. In conclusion, the contribution of nitric oxide to basal vessel tone was not impaired in the heart, skeletal muscle and kidney in SHR. Antagonism of ETA receptors caused similar haemodynamic responses in both rat strains in these organs. Furthermore, NOS inhibition, but not ETA blockade, blunted the expected ACh-induced vasodilation in the heart and kidney in WKY rats, but not in skeletal muscle in both strains.     

Nonpeptide endothelin receptor antagonists. XI. Pharmacological characterization of SB 234551, a high-affinity and selective nonpeptide ETA receptor antagonist

The present study describes the pharmacological profile of ((E)-alpha-[[1-butyl-5-[2-[(2-carboxyphenyl)methoxy]-4-methoxy-phenyl ]-1H-pyrazol-4-yl]methlene]-6-methoxy-1,3-benzodioxole-5-propanoic acid) (SB 234551), a high-affinity, nonpeptide endothelin type A (ETA)-selective receptor antagonist. In human cloned ETA and endothelin type B (ETB) receptors, SB 234551 produced a concentration-dependent displacement of [125I]-endothelin-1 with Ki values of 0.13 and 500 nM, respectively. SB 234551 elicited concentration-dependent, rightward competitive shifts in the endothelin-1 concentration-response curves in isolated rat aorta and isolated human pulmonary artery (ETA receptor-mediated vascular contraction) with Kb values of 1.9 and 1.0 nM, respectively. SB 234551 antagonized ETB receptor-mediated vasoconstriction in the isolated rabbit pulmonary artery, as demonstrated by concentration-dependent, rightward shifts in the sarafotoxin S6c concentration-response curves (Kb = 555 nM). SB 234551 produced weak functional inhibition of sarafotoxin S6c-mediated endothelium-dependent relaxation (IC50 = 7 microM). SB 234551 (10 microM) had no significant effect against contraction produced by several other vasoactive agents and did not significantly influence radioligand binding to a number of diverse receptors. SB 234551 (0. 1-1.0 mg/kg i.v.) dose-dependently inhibited the pressor response to exogenous endothelin-1 in conscious rats. In vivo pharmacokinetic analysis in the rat demonstrated that SB 234551 was rapidly absorbed from the GI tract with a bioavailability of 30%. SB 234551 had a plasma half-life of 125 min and a systemic clearance of 25.0 ml/min/kg. The present study demonstrates that SB 234551 is an antagonist with high affinity for the ETA receptor, while sparing the ETB receptor. SB 234551 is a new pharmacological tool that should assist in the elucidation of the role of endothelin in pathophysiology.     

Expression of a functional tagged human thyrotropin receptor in HeLa cells using recombinant vaccinia virus

The aim of the present study was to assess the prognostic relevance of relatives' interactive behaviour towards the patient, as covered by the Münster Family Interview (MFI), to the further course of the schizophrenic illness. The MFI is a family interview (of the whole family, including the patient) designed to record the emotional family atmosphere based on the concept of expressed emotion (EE). The ratings take place directly after the interview on five scales (criticism, hostility, overinvolvement, resignation and warmth), the resignation scale being added to the 'classic' EE scales. Ninety-nine families of outpatients diagnosed with schizophrenia according to the DSM-III were examined with the MFI during a home visit. The patients were seen 1 and 2 years after the first examination. The target criteria selected for the prognostic significance of the interaction measurements were: rehospitalisation within 2 years; extent of symptoms after 1 year, and psychosocial skills after 1 year. The significance of the interaction dimensions was verified in regression models. The control variable used in the regression models was the Strauss-Carpenter scale. Regression models were produced for the total group and for a subgroup of moderately ill patients. All target criteria yielded serviceable prediction models. The most important variable for prediction was the control variable, the Strauss-Carpenter scale. However the interaction variables made additional contributions to the prognosis, especially in the subgroup of moderately ill patients. The best MFI scale for all the outcome criteria was resignation; criticism predicted only the symptomatology, and emotional overinvolvement the level of social functioning after 1 year. In conclusion, practical work with families of schizophrenic patients should emphasise the protective function of relatives towards patients more strongly.     

ETA receptor-mediated role of endothelin in the kidney of DOCA-salt hypertensive rats

Renal effects of FR139317, an endothelin ETA receptor antagonist, were examined using anesthetized normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The intravenous bolus injection of FR139317 (10 mg/kg) produced a slight decrease in mean blood pressure (MAP; -13%) in the control rats and this hypotension was accompanied by a moderate renal vasodilation (renal vascular resistance: RVR; -12%). In the DOCA-salt hypertensive rat, FR139317 had a more pronounced hypotensive effect (MAP; -26%) accompanied by a potent renal vasodilation (RVR; -33%). FR139317 significantly increased renal blood flow only in the DOCA-salt rats. In contrast, FR139317 produced a significant decrease in urine flow and urinary sodium excretion only in control rats. Northern blot analysis revealed that the renal prepro endothelin-1 (ET-1) mRNA level was significantly increased in DOCA-salt hypertensive rats. Thus, it seems likely that endogenous ET-1 is responsible for the maintenance of DOCA-salt-induced hypertension. We also suggest that at least in part, ET-1 and ETA receptors are involved in renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-1 production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension.     

Suppressive effects of the endothelin receptor (ETA) antagonist BQ-123 on ET-1-induced reduction of lipoprotein lipase activity in 3T3-L1 adipocytes

Endothelin (ET)-1 reduced heparin-releasable lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes in a concentration-dependent manner. However, a selective ETB receptor agonist, [Ala1,3,11,15]ET-1, did not act like ET-1. The ET-1-induced decrease in LPL activity was suppressed by a selective ETA receptor antagonist, BQ-123: the concentration-response curve for the ET-1 reduction of LPL activity was shifted to the right in the presence of BQ-123 in a concentration-dependent manner. This antagonistic effect of BQ-123 clarifies that the ETA receptor is responsible for the ET-1-induced reduction of LPL activity in 3T3-L1 adipocytes, which suggests that there is therapeutic potential for ETA antagonists in LPL-related lipoprotein disorders.     

Expression of substance P (NK1) receptor mRNA in human nose

A capillary zone electrophoretic method (CZE) was developed using an uncoated fused silica capillary for the separation and determination of the main tropane alkaloids. The applicability of the developed method for analysis of plant samples was examined by analyzing samples of transgenic Egyptian henbane Hyoscyamus muticus (L.) plants. A simple 40 mM phosphate buffer at pH 7.8 using a voltage of 20 kV was found the best for this purpose. The main tropane alkaloids, atropine and scopolamine as well as nor-(-)-scopolamine, and tropic acid, the precursor of tropane alkaloids, could be separated in less than 13 min. The linear concentration range for atropine was 5.00-140 microg ml(-1), for scopolamine 7.50-210 microg ml(-1) and for tropic acid 2.50-70.0 microg ml(-1).     

Evidence for a tyrosine kinase-dependent activation of the adenylyl Cyclase/PKA cascade downstream from the G-protein-linked endothelin ETA receptor in vascular smooth muscle

Body retinoids are stored in the lipid droplets of hepatic stellate (Ito) cells. In chronic liver disease, the stellate cells differentiate into myofibroblast-like cells, a process whereby they lose their retinoid-containing lipid droplets. We studied the relation between liver retinoid content, the number of lipid droplets per stellate cell, and the number of stellate cells per mm2 in human alcoholic liver disease. Semithin sections of liver biopsies from normal subjects and patients with early (steatosis, inflammation, and mild fibrosis) and late (cirrhosis and cirrhosis with acute alcoholic hepatitis) alcoholic liver disease were morphometrically evaluated. Liver retinoid content was determined by HPLC. In normal patients, liver retinoid content was 901 +/- 213 IU/g of liver (mean +/- SEM). There was a decrease in liver retinoid content in early alcoholic liver disease (409 +/- 50 IU/g) and a further reduction in cirrhosis (153 +/- 50 IU/g). In patients with acute alcoholic hepatitis, retinoid content was strikingly low (5.2 +/- 1.8 IU/g). There was a progressive decrease in the number of stellate cells per mm2 associated with progressive liver damage. We found a fair correlation between the number of stellate cells per mm2 and liver retinoid content in all patient groups (overall correlation: 0.71). In normal subjects, the mean number of lipid droplets per stellate cell was 7.4 +/- 0.7. In patients with early alcoholic liver disease and in patients with alcoholic cirrhosis, this value was increased to 13.6 +/- 0.8 and 10.4 +/- 2.0, respectively. In patients with acute alcoholic hepatitis, only a few lipid droplets were present (4.2 +/- 0.5). There was a good correlation between liver retinoid content and mean number of lipid droplets in normal patients (r = 0.58). In alcoholic cirrhosis, however, correlation was poor (r = 0.34). In early alcoholic liver disease, the correlation was absent (r = 0.004). In conclusion, the major finding of our study is that the correlation between the mean number of lipid droplets per stellate cell and liver retinoid content varies according to the hepatic pathology considered. Marked lipid droplet accumulation occurs in stellate cells in early alcoholic liver disease and, to a lesser extent, in alcoholic cirrhosis, but there is no correlation between the mean number of lipid droplets per stellate cell and liver retinoid content. Therefore, not retinoids but probably lipids are responsible for the accumulation of lipid droplets. We also find that there is a fair correlation between the number of stellate cells per mm2 and liver retinoid content in all patient groups. Finally, we confirm the decrease in hepatic retinoid content that occurs in alcoholic liver disease in humans, even at the early stages of the disease.     

Expression of a functional anaphylatoxin C3a receptor by astrocytes

Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand-binding studies, functional responses, and RNA analysis by RT-PCR. Uptake of 125I-C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High-affinity binding sites were abundantly expressed (20,000-80,000 sites per cell) with an estimated K(D) of 1-2 nM. Low-affinity binding sites with a K(D) of 209 nM were largely expressed (n > or = 4 x 10(6) sites per cell) and probably did not reflect a receptor-mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT-PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti-C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca2+] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C-terminus Arg considerably decreased the [Ca2+] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.     

Evidence for heterogeneity of endothelin receptor distribution in human coronary artery

1. The receptors mediating endothelin-evoked contraction of human coronary artery have been investigated in isolated segments of the left anterior descending coronary artery (LAD). 2. Endothelin-1 (ET-1) was 10 times more potent in distal than in proximal segments but the potency ratio between ET-1 and ET-3 (endothelin-3) was similar and close to 100 in any segment of the artery. 3. BQ-123, an ETA receptor antagonist, competitively antagonized the response to ET-1 of distal segments (pA2 equal to 7.47). In the proximal segments, part of the contractile response was BQ123 sensitive, but the antagonism was non-competitive. In both groups of segments, the response to ET-3 could be completely blocked by BQ-123. 4. These observations indicate that ETA receptors mediate the contractile response to ET-1 in distal, pre-resistant coronary arteries, but that other ET receptors are also involved in the contractile response of proximal segments.     

Endothelin-1-induced in vitro cerebral venoconstriction is mediated by endothelin ETA receptors

The in vitro effects of endothelin-1 on cerebral veins were studied using cylindrical segments, 5 mm long, from dog pial veins. Isometric responses to endothelin-1 (10(-12)-10(-7) M) and to the endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21), 10(-12) -10(-7) M), were recorded in veins under control conditions and pretreated with the endothelin ET(A) receptor antagonist, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp), 10(-8) -10(-5) M), and the endothelin ETB receptor antagonist, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(me thoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium, 10(-6) and 10(-5) M). The response to endothelin-1 was also recorded in veins pretreated with the nitric oxide synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), or the cyclooxygenase inhibitor, meclofenamate (10(-5) M), and in veins without endothelium or placed in medium without Ca2+ but with EDTA (0.1 mM). In control veins, endothelin-1 produced a concentration-dependent contraction (EC50 = 2.0 x 10(-10) M; maximal contraction = 113 +/- 6 mg) and IRL 1620 induced no effects or a small contraction only with high concentrations (10(-8) - 10(-6) M) (EC50 = 1.5 x 10 (-8) M; maximal contraction = 9 +/- 3 mg). BQ-123 shifted the response to endothelin-1 to the right in a parallel, concentration-dependent way, whereas BQ-788, L-NAME or meclofenamate did not modify the response to endothelin-1. Compared with the control, veins in a medium without Ca2+ had similar EC50 values, but a lower maximal contraction induced by endothelin-1 (57 +/- 10 mg, P < 0.05), and veins without endothelium exhibited similar EC50 values. Thus, endothelin-1 produces marked cerebral venoconstriction that could be mainly mediated by activation of endothelin ETA receptors, may be dependent on extracellular Ca2+, and may be independent of endothelium, nitric oxide and prostanoids.     

The effect of the ETA receptor antagonist (CI-1020) in rats with established hypoxic pulmonary hypertension

ETA receptor antagonists have previously been shown to prevent the development of pulmonary hypertension induced by chronic hypoxia in the rat. Clinically, however, patients present with already established pulmonary hypertension. We have investigated the effects of the ETA receptor antagonist CI-1020 in rats previously adapted to chronic hypoxia. Two protocols were followed. Rats (n=32) were divided into two batches of four groups: normoxic controls in air for 10 days (NC10), chronic hypoxic controls in hypoxia for 10 days (CHC10), chronic hypoxic vehicle treated in hypoxia for 20 days (CHV20) and chronic hypoxic drug treated in hypoxia for 20 days (CHT20). Ten days after the onset of hypoxia, oral treatment with drug (40 mg/kg per day) or vehicle was started. Animal weight, ratio of right ventricular weight to left ventricular weight including septum (RV/LV+S) and percentage of double elastic lamina (DEL) were determined. In the second study, 12 rats were divided into three groups; normoxic controls in air for 20 days (NC20), (CHV20) and (CHT20). After 10 days hypoxia, oral treatment with drug (40 mg/kg per day) or vehicle was started. Isolated perfused lung preparations were then used to determine pulmonary artery pressure and pulmonary vascular resistance. Treatment with CI-1020 reduced the increase in RV/LV+S and the percentage DEL induced by chronic hypoxia and significantly lowered the increase in pulmonary resistance in isolated perfused lungs from chronically hypoxic animals. These results suggest that CI-1020 could have an important role in the treatment and reversal of established pulmonary vascular remodelling.     

Discovery of IRL 3461: a novel and potent endothelin antagonist with balanced ETA/ETB affinity

IRL 3461, N-butanesulfonyl-[N-(3,5-dimethylbenzoyl)-N-methyl-3-[4-(5-+ ++isoxazolyl) -phenyl]-alanyl]-(L)-valineamide, a potent and bifunctional (ETA + ETB) [Ki(ETA) = 1.8 nM, Ki(ETB) = 1.2 nM] antagonist was discovered by structural modification of IRL 2500, an ETB selective antagonist. IRL 3461 was found to be stable on incubation with human, rat, mouse, and guinea pig plasmas.     

Activation of endothelin ETA receptors induces phosphorylation of alpha1b-adrenoreceptors in Rat-1 fibroblasts

The effect of endothelin-1 on the phosphorylation of alpha1b-adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal alpha1b-adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC50 approximately 1 nM), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ETA receptors. Endothelin-1-induced alpha1b-adrenoreceptor phosphorylation was attenuated by staurosporine or genistein and essentially abolished when both inhibitors were used together. The effect of norepinephrine was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used together. These data suggest the participation of protein kinase C and tyrosine kinase(s) in endothelin-1-induced receptor phosphorylation. However, phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not of phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by endothelin, could act in a step previous to receptor phosphorylation. The effect of endothelin-1 on alpha1b-adrenoreceptor phosphorylation was not mediated through pertussis toxin-sensitive G proteins. Calcium mobilization induced by norepinephrine was diminished by endothelin-1. Norepinephrine and endothelin-1 increased [35S]GTPgammaS binding to control membranes. The effect of norepinephrine was abolished in membranes obtained from cells pretreated with endothelin-1. Interestingly, genistein plus staurosporine inhibited this effect of the endothelial peptide. Endothelin-1 did not induce alpha1b-adrenoreceptor internalization. Our data indicate that activation of ETA receptors by endothelin-1 induces alpha1b-adrenoreceptor phosphorylation and alters G protein coupling.     

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 3. Discovery of a potent, 2-nonaryl, highly selective ETA antagonist (A-216546)

Previously we have reported the discovery of ABT-627 (1, A-147627, active enantiomer of A-127722), a 2,4-diaryl substituted pyrrolidine-3-carboxylic acid based endothelin receptor-A antagonist. This compound binds to the ETA receptor with an affinity (Ki) of 0. 034 nM and with a 2000-fold selectivity for the ETA receptor versus the ETB receptor. We have expanded our structure-activity studies in this series, in an attempt to further increase the ETA selectivity. When the p-anisyl group of 1 was replaced by an n-pentyl group, the resultant antagonist 3 exhibited substantially increased ETB/ETA activity ratio, but a decreased ETA affinity. Structure-activity studies revealed that substitution and geometry of this alkyl group, and substitution on the benzodioxolyl ring, are important in optimizing this series of highly ETA selective antagonists. In particular, the combination of a (E)-2,2-dimethyl-3-pentenyl group and a 7-methoxy-1,3-benzodioxol-5-yl group provided hydrophobic compound 10b with subnanomolar affinity for human ETA receptor subtype and with an ETB/ETA activity ratio of over 130000. Meanwhile, synthetic efforts en route to olefinic compounds led to the discovery that 2-pyridylethyl (9o) and 2-(2-oxopyrrolidinyl)ethyl (9u) replacement of the p-anisyl group of 1yielded very hydrophilic ETA antagonists with potency and selectivity equal to those of 10b. On the basis of overall superior affinity, high selectivity for the ETA receptor (Ki, 0.46 nM for ETA and 13000 nM for ETB), and good oral bioavailability (48% in rats), A-216546 (10a) was selected as a potential clinical backup for 1.