Studies on coronary arteriopathy in dogs following administration of CI-1020, an endothelin A receptor antagonist.

A selective nonpeptide endothelin A (ETA) receptor antagonist, CI-1020, was administered to beagle dogs intravenously (i.v.) for 4 hours to 4 weeks. One animal/sex received CI-1020 at 1 mg/kg/hr intravenously for 4, 8, or 24 hours to investigate onset of arteriopathy. Control animals (1/sex) received the vehicle only. To determine reversibility of arteriopathy, 8 dogs/sex were given CI-1020 at 1 mg/kg/hr for 4 days. Two dogs/sex were sacrificed 1, 3, 8, and 29 days following cessation of infusion. Lesion development with prolonged exposure was investigated in 1 male dog. It was given CI-1020 by i.v. bolus at 120 mg/kg/day for 4 weeks and Monastral blue dye was administered i.v. to facilitate localization of vascular lesions. Coronary blood flow was determined in 4 dogs infused with CI-1020 at 0.3, 3, and 30 mg/kg for one hour at each dose. Macroscopically, hemorrhage or blue discoloration of Monastral blue was noted in the extramural coronary arteries along the coronary groove and atrium. Histologically, the earliest coronary changes were noted in animals sacrificed after 24 hours of treatment and characterized by medial hemorrhage and necrosis with a few infiltrating neutrophils. In the reversibility study, incidence and severity of arteriopathy was dependent on time of sacrifice following cessation of infusion. Acute necrotizing inflammation of arteries was present in all animals (n = 4) on day 1 postinfusion, whereas on day 8 postinfusion, lesions characterized by medial small pockets of trapped red cells, cell debris, and adventitial thickening were seen in 1 dog/sex. By day 29 postinfusion, coronary arteries were similar to controls. In the dog given daily i.v. bolus injections of CI-1020 for 4 weeks, arterial inflammatory lesions varied from acute to chronic, although most lesions were considered chronic active. Monastral blue pigments were noted in the wall of most arteries with chronic or chronic active lesions. Acute lesions were similar to those noted in day 1 postinfusion of the reversibility study. Medial smooth muscle necrosis and/or fibrosis with mixed inflammatory cell infiltrates characterized chronic or chronic active lesions. Smooth muscle proliferation and migration into the intima were also noted. There were no significant changes in coronary blood flow, coronary vascular resistance, or mean arterial blood pressure following CI-1020 infusion for 3 hours. In the 24-hour infusion study, plasma endothelin 1 (ET-1) levels were mildly elevated (1.5-4 fold) during CI-1020 infusion when compared to either pretest or control values. These results indicate that administration of endothelin antagonist (CI-1020) to dogs was associated with development of coronary arteriopathy, which was completely resolved within 29 days following cessation of treatment. With prolonged (4-week) CI-1020 treatment, arterial lesions at varying stages of development (acute, chronic active, chronic) were seen, suggesting that tolerance to treatment (up to 4 weeks) does not occur.

The endothelin-A-receptor antagonist PD 180988 (CI-1034) selectively reverses the pulmonary vasoconstrictor response to hypoxia in the lamb.

Endothelin-1 (ET-1) is assigned a mediator role in the constrictor response of the pulmonary vasculature to hypoxia. Accordingly, a recently developed endothelin-A (ETA) antagonist, PD180988, was tested in the chronically instrumented newborn lamb to verify this possibility and, at the same time, to study a potential new treatment for pulmonary hypertension (PH). PD180988, given by infusion after a priming bolus, had an insignificant effect on the pulmonary circulation under normoxia, while it reversed the sustained pulmonary constriction caused by hypoxia. No appreciable change was noted under either experimental condition in the systemic circulation and cardiac contractility. We conclude that PD180988 is a selective inhibitor of hypoxic pulmonary vasoconstriction and lends itself to therapeutic use in infants.

Butenolide endothelin antagonists with improved aqueous solubility.

Continued development around our ETA-selective endothelin (ET) antagonist 1 (CI-1020) has led to the synthesis of analogues with improved aqueous solubility profiles. Poor solubility characteristics displayed by 1 required a complex buffered formulation in order to conduct iv studies. To overcome the use of specific iv formulations for preclinical studies on additional drug candidates, analogues with improved aqueous solubility were desired. Several analogues were synthesized with substitution patterns that allowed for the formation of either acid or base addition salts. These derivatives had dramatically improved aqueous solubility. In addition, these analogues retained equivalent or improved ETA receptor selectivity and antagonist potency, versus 1, both in vitro and in vivo. Compound 29, which contains as a substituent the sodium salt of a sulfonic acid, has an ETA IC50 = 0.38 nM, ETA selectivity of 4200-fold, and ETA functional activity of KB = 7.8, all of which are similar or superior to those of 1. Compound 29 also has vastly superior aqueous solubility and solubility duration, compared to 1. Furthermore, 29 after iv infusion displays improved activity to 1 in preventing acute hypoxia-induced pulmonary hypertension in rats with an ED50 = 0.3 microg/kg/h.

PD 142893, SB 209670, and BQ 788 selectively antagonize vascular endothelial versus vascular smooth muscle ET(B)-receptor activity in the rat.

The purpose of this study was to determine whether vascular endothelial and vascular smooth-muscle endothelin ET(B) receptors could be quantitatively differentiated by PD 142893 (PD), SB 209670 (SB), and BQ 788 (BQ) in the same species by using closely matched experimental conditions. The isolated perfused rat kidney (vascular smooth muscle) and isolated perfused rat mesentery (vascular endothelium) were challenged with increasing bolus doses of sarafotoxin S6c in the absence and presence of antagonist. PD, SB, and BQ produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the kidney. PD and SB also produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the mesentery. In contrast, BQ produced an insurmountable antagonism. Schild-derived pA2 values for PD and SB were significantly greater for inhibiting endothelial versus smooth-muscle ET(B) receptors. Furthermore, PD and SB differed in their relative potency between the two assays. Because BQ produced an insurmountable antagonism in the mesentery, it was not possible quantitatively to compare the antagonist activity in the two assays. These results indicate that PD, SB, and BQ selectively antagonize endothelial ET(B)-receptor activity over smooth-muscle ET(B)-receptor activity.

Endothelin receptor antagonists: synthesis and structure-activity relationships of substituted benzothiazine-1,1-dioxides.

The development of benzothiazine-1,1-dioxide derivatives as a new structural class of potent endothelin receptor antagonists is described. Structure-activity relationships (SAR) revealed that PD164800 (1) is a potent antagonist of the ETA receptor subtype.

Endothelin B receptors on human endothelial and smooth-muscle cells show equivalent binding pharmacology.

We have described the pharmacologic profiles of endothelin B receptors in human endothelial cells and vascular and nonvascular smooth-muscle cells. First, by amplifying endothelin B receptor numbers through the use of phosphoramidon and intact cell-binding techniques, we demonstrated the presence of these receptors in human umbilical vein endothelial cells (100% endothelin B receptors), human aortic smooth-muscle cells (22% endothelin B, 78% endothelin A receptors), and human bronchial smooth-muscle cells (55% endothelin B, 45% endothelin A receptors) by using [125I]-endothelin-1 radioligand binding. The typical binding profiles of the endothelin B receptors were established through competition binding curve analysis with endothelin-1, endothelin-3, sarafotoxin 6c, and the endothelin A receptor-selective antagonist BQ-123. In the presence of BQ-123, a diverse group of antagonists, including PD 142893, BQ-788, SB 209670, and Ro 47-0203, were used to probe for binding differences indicative of multiple endothelin B-receptor subtypes. The results indicate a rank order of potency for the antagonists of BQ-788 > SB 209670 > PD 142893 > Ro 47-0203 for each cell line, and that between any of these human cell lines, measurements of [125I]-endothelin-1-binding antagonism for each of the four test compounds differed by less than twofold. Although this study cannot discount the possibility of more than one endothelin B-receptor subtype in humans, it does indicate that these tissues express receptors that show equivalent binding pharmacology.

Assessment of endothelin receptor subtype-mediated increases of [Ca2+]i in distinct rat cell types using fluorimetric imaging.

Activation of endothelin (ET) receptor subtypes by various agonists causes an increase in [Ca2+]i in different cell types. This effect can be readily monitored in a 96-well plate format by detecting 1-s fluorescence changes of cell-permeant, Ca(2+)-sensitive dyes (e.g., Calcium Green-1 AM) using a fluorimetric imaging plate reader. This device was used to assess the ET receptor subtypes in primary cultures of rat mixed neocortical neuronal/glial cells and aortic smooth-muscle cells. Pharmacologic experiments with several ET receptor agonists and antagonists indicated that the ETA receptor subtype was functionally responsive in the smooth-muscle cells and that the ETB receptor subtype had a similar role in the mixed neuronal/glial cells.

Efficacy of CI-1020, an endothelin-A receptor antagonist, in hypoxic pulmonary hypertension.

We previously showed that CI-1020, an endothelin (ET)-A-selective receptor antagonist, dose-dependently blocked acute hypoxic pulmonary hypertension (PH) in rats. In this study we show that CI-1020 can reverse existing PH and prevent progression of right ventricular hypertrophy (RVH) in rats exposed to chronic hypoxia. Male Sprague-Dawley rats were exposed to 20 days of hypoxia (10% O2) with CI-1020 treatment (20 or 40 mg/kg/day) starting on day 10. On day 20 of hypoxia, the rats were instrumented under anesthesia with a pulmonary artery cannula and allowed to recover to consciousness before measurement of mean pulmonary arterial pressure (MPAP). Blood samples were then collected for plasma ET-1 measurements, the rats killed, and their hearts dissected, dried, and weighed. RV/LV + septum ratio (g/g) was used as an index of RVH (RVHi). Normoxic rats and rats exposed to hypoxia for only 10 days were also evaluated as controls. Normoxic rats had MPAPs of 13 +/- 1 mm Hg, plasma ET-1 levels of 2.1 +/- 0.1 pg/ml, and an average RVHi of 0.29 +/- 0.03. Rats exposed to 10 or 20 days of hypoxia had MPAPs of 33 +/- 2 and 44 +/- 0 mm Hg, plasma ET-1 levels of 4.2 +/- 0.8 and 4.6 +/- 0.8 pg/ml, and average RVHis of 0.47 +/- 0.05 and 0.52 +/- 0.03, respectively. In comparison, rats treated with CI-1020 had MPAPs that were 37% (20 mg/kg/day) and 44% (40 mg/kg/day) lower than untreated 20-day hypoxic rats. Furthermore, rats dosed with 40 mg/kg/day of CI-1020 had MPAPs that were significantly lower (24%) than control 10-day hypoxic rats, indicating a significant reversal of PH. Along with this reversal in PH, their average RVHi was 23% lower (p < 0.05) relative to untreated 20-day hypoxic rats.

Novel selective quinazoline inhibitors of endothelin converting enzyme-1.

PD 069185 is a highly selective and structurally novel inhibitor of endothelin converting enzyme-1 (ECE-1). PD 069185 is a trisubstituted quinazoline with an IC50 value of 0.9 +/- 0.1 microns for inhibition of human ECE-1 from the solubilized membrane fraction of CHO cells stably transfected with human ECE-1 cDNA. Kinetic analysis revealed that PD 069185 is best fit with a competitive inhibition model with a Ki value of 1.1 +/- 0.1 microns and binds in a reversible manner. The closely related enzyme, ECE-2, is not inhibited at up to 100 microns PD 069185. In addition, PD 069185 at 200-300 microns has little effect on other metalloproteases, such as neutral endopeptidase 24.11, stromelysin, gelatinase A, and collagenase, showing a high ECE-1 specificity. Data are also presented to show that this series of inhibitors are effective in inhibiting ECE-1 in intact cells and in attenuating the increase in perfusion pressure induced by big ET-1 in isolated rat mesentery. These non-peptidic ECE-1 inhibitors should serve as a valuable tool to study the pathophysiological role of endothelin and the therapeutic potential of ECE-1 inhibitors.

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.

Role for endothelin-1 in angiotensin II-mediated hypertension.

Experiments in cultured vascular smooth muscle cells have shown that angiotensin II (Ang II) stimulates expression of endothelin-1. We sought to examine role of endothelin-1 in the effects of Ang II in vivo. Ang II infusion in rats (0.7 mg/kg per day for 5 days) was associated with marked increases in vascular smooth muscle endothelin-1 levels, as assessed by immunostaining. Administration of the selective endothelin type A (ET(A)) receptor antagonist PD 155080 (50 mg/kg per day) abrogated the hypertensive response to a 5-day infusion of Ang II (0.7 mg/kg per day), as did losartan (25 mg/kg per day). ET(A) receptor blockade during Ang II-mediated hypertension was associated with marked elevations of plasma endothelin-1 levels. Ang II-mediated hypertension was associated with heightened vascular responsiveness to a variety of vasoconstrictor agents except endothelin-1. Blockade of ET(A) receptor invariably corrected this vasoconstrictor hyperresponsiveness. We conclude that some of the vascular effects of Ang II thought to be unique to this hormone are likely mediated by endothelin-1.

Design of a potent combined pseudopeptide endothelin-A/endothelin-B receptor antagonist, Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of its pharmacokinetic and spectral properties.

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

Cardiopulmonary indications for endothelin receptor antagonists: review of recent efficacy trials.

Recent clinical and experimental animal trials indicate that endogenously produced endothelin-1 (ET-1) contributes to the abnormal systemic and pulmonary vascular resistance associated with congestive heart failure (CHF) and pulmonary hypertension (PH). In experimental CHF, the chronic blockade of ET-1 actions by ET receptor antagonists clearly improves haemodynamic status, and improves cardiac structure and survival. The latter is based on limited results. In experimental PH there are consistent reports of prevention and reversal of PH, pulmonary vascular remodelling and right ventricular hypertrophy, independent of the inciting mechanisms. These results in experimental animals illustrate the potential efficacy of the ET receptor antagonists in future clinical trials. With five ET receptor antagonists in clinical development, and more on the way, their potential will soon be realised.

Structure-activity relationships in a series of orally active gamma-hydroxy butenolide endothelin antagonists.

The design of potent and selective non-peptide antagonists of endothelin-1 (ET-1) and its related isopeptides are important tools defining the role of ET in human diseases. In this report we will describe the detailed structure-activity relationship (SAR) studies that led to the discovery of a potent series of butenolide ETA selective antagonists. Starting from a micromolar screening hit, PD012527, use of Topliss decision tree analysis led to the discovery of the nanomolar ET(A) selective antagonist PD155080. Further structural modifications around the butenolide ring led directly to the subnanomolar ETA selective antagonist PD156707, IC50's = 0.3 (ET(A)) and 780 nM (ET(B)). This series of compounds exhibited functional activity exemplified by PD156707. This derivative inhibited the ETA receptor mediated release of arachidonic acid from rabbit renal artery vascular smooth muscle cells with an IC50 = 1.1 nM and also inhibited the ET-1 induced contraction of rabbit femoral artery rings (ETA mediated) with a pA2 = 7.6. PD156707 also displayed in vivo functional activity inhibiting the hemodynamic responses due to exogenous administration of ET-1 in rats in a dose dependent fashion. Evidence for the pH dependence of the open and closed tautomerization forms of PD156707 was demonstrated by an NMR study. X-ray crystallographic analysis of the closed butenolide form of PD156707 shows the benzylic group located on the same side of the butenolide ring as the gamma-hydroxyl and the remaining two phenyl groups on the butenolide ring essentially orthogonal to the butenolide ring. Pharmacokinetic parameters for PD156707 in dogs are also presented.

Structure-activity relationships of the potent combined endothelin-A/endothelin-B receptor antagonist Ac-DDip16-Leu-Asp-Ile-Ile-Trp21: development of endothelin-B receptor selective antagonists.

The endothelins (ETs) are a family of bicyclic 21-amino acid-containing peptides that are highly potent and prolonged vasoconstrictors. The discovery of potent ET antagonists will facilitate the understanding of the physiological and/or pathophysiological role of ET. Structure-activity studies have revealed the importance of the C-terminal hexapeptide (residues 16-21) of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21) to the development of potent antagonists at both receptor subtypes (ETA and ETB). In particular, it has been shown that Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (Dip = 3,3-diphenylalanine) has low nanomolar affinity for the two endothelin receptor subtypes and is a functional antagonist of ET activity, both in vitro and in vivo at both receptors. Herein, we will describe the structure-activity relationships of Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) with a particular emphasis on modifications that lead to enhanced receptor affinity and/or individual receptor subtype selectivity. In particular, we will demonstrate how we utilized PD 142893 to develop ETB receptor selective ligands and the pharmacological differences that exist between species ETB receptors with respect to their affinity for C-terminal hexapeptide antagonists.

Pharmacological characterization of PD 156707, an orally active ETA receptor antagonist.

We describe the pharmacological characteristics of PD 156707 (sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5- trimethoxy-benzyl)-but-2-enoate), a potent, orally active, nonpeptide antagonist of the endothelin A (ETA) receptor subtype. PD 156707 was designed on the basis of a compound identified by screening the Parke-Davis chemical library. PD 156707 is highly selective for the ETA receptor (ETAR) and inhibits the binding of [125I]-ET-1 to cloned human ETAR and ETBR with Ki values of 0.17 and 133.8 nM, respectively. PD 156707 antagonizes ET-1-stimulated phosphoinositide hydrolysis in Ltk- cells expressing cloned human ETAR with an IC50 value of 2.4 nM. PD 156707 inhibits vasoconstriction in isolated blood vessels mediated by ETAR (rabbit femoral artery) and ETBR (rabbit pulmonary artery) with pA2 values of 7.5 and 4.7, respectively. PD 156707 administered orally to rats blocked subsequent ETAR-mediated pressor responses in vivo but had no effect on ETBR-mediated dilator responses. As a potent and orally active ETA-selective antagonist, PD 156707 will be useful in defining the physiological and pathological roles of ETAR.

Structure-activity relationships of a novel series of orally active nonpeptide ETA and ETA/B endothelin receptor-selective antagonists.

The development of nonpeptide, low molecular weight antagonists with high potency, oral activity, and selectivity is an important objective to adequately define the potential role of endothelin (ET) and its isopeptides in human diseases. This report describes the structure-activity relationships, ETA/ETB selectivity, and pharmacokinetics of the PD 155080 and PD 156707 series of orally active nonpeptide ET receptor-selective antagonists. Modification of the substituents around the butenolide ring has led to compounds with differing selectivity for human ETA and ETB receptors. Thus, compounds with increased lipophilicity at R2 show increased ETB affinity and a more balanced ETA/ETB profile. For example, the 4-O-n-pentyl analogue of PD 156707 is a potent competitive inhibitor of [125I]ET-1 and [125I]ET-3 binding to human cloned ETA and ETB receptors, with IC50s of 0.8 nM and 44 nM, respectively. Pharmacokinetic properties can also be significantly influenced by structural modifications at the R2 group. The pharmacokinetics of PD 155719, PD 155080, and PD 156707 were compared in male Wistar rats after a 15 mg/kg intravenous or oral gavage dose (three animals per dose). Plasma concentrations were determined by a specific HPLC assay. Oral bioavailability ranged from less than 5% for PD 155719 to 41% for PD 156707 and 87% for PD 155080.

Effects of selective endothelin antagonists on the hemodynamic response to cyclosporin A.

Cyclosporin A (CsA) treatment is associated with hypertension and renal dysfunction. Increased circulating endothelin (ET) has been implicated in this renal dysfunction, which is secondary to renal vasoconstriction and decreases in RBF. The effects of the selective blockade of ETA receptors or the combined blockade of ETA and ETB receptors on the acute hemodynamic response to CsA in anesthetized rats were examined. Rats were pretreated with vehicle, BQ-123 (selective ETA receptor antagonist; 0.6 micron/kg per minute), or BQ-123 and PD 142893 (combined ETA/ETB receptor antagonist; 0.6 micron/kg per minute) to achieve both ETA and ETB receptor blockade. After a 10-min pretreatment, CsA (20 mg/kg over 10 min) was administered; mean arterial blood pressure, RBF, and iliac blood flow were monitored continuously. Hemodynamic responses to exogenous endothelin-1 (ET-1, 0.3 and 1 nmol/kg) with and without antagonist pretreatment were measured in separate groups to demonstrate effective receptor blockade. CsA elevated blood pressure 17 to 20% in all three groups; renal resistance maximally increased 23, 20, and 23% in vehicle, BQ-123, and BQ-123 and PD 142893 pretreated groups, respectively. In contrast, the combination of BQ-123 and PD 142893 blocked systemic pressor responses to 0.3 and 1 nmol of ET-1 approximately 50 and 37%, respectively; changes in renal resistance were blocked 81 and 89%, respectively. In conclusion, the elevation in systemic blood pressure and the renal vasoconstrictor activity of CsA do not appear to be mediated through ETA or ETB receptors.

ETA and ETB receptors coexist on rabbit pulmonary artery vascular smooth muscle mediating contraction.

The possibility that both ETA and ETB endothelin receptor subtypes could mediate contractile activity in the same tissue was investigated in isolated, endothelium denuded rabbit pulmonary arteries. The ETB selective agonist, sarafotoxin 6c (S6c), produced potent contractile activity, equal to the non-selective ETA and ETB receptor agonist endothelin-1 (ET-1), indicating a contractile role for ETB receptors in this tissue. In addition BQ-123 (10.0 microM), the ETA selective antagonist, was only partially effective in blocking ET-1 induced contractions further indicating a contractile role for ETB receptors. However, the partial blockade by BQ-123 suggested a possible contractile role for ETA receptors. To address this possibility, ETB receptors were desensitized with a 30 minute pretreatment of S6c (0.01 microM). Under these conditions, we were able to demonstrate full ET-1 contractile activity that was now sensitive to blockade by BQ-123. The coexistence of both ETA and ETB receptors was confirmed through receptor binding experiments indicating 40/60 ratio, respectively. We conclude that 1) both ETA and ETB receptors coexist on vascular smooth muscle of rabbit pulmonary artery, 2) activation of either receptors subtype results in contraction, and 3) prolong activation of the ETB receptor subtype produces tachyphylaxis preventing further activation by S6c or ET-1.

Why big endothelin-1 lacks a vasodilator response.

The biphasic arterial blood pressure response to endothelin-1 (ET-1) results from a transient decrease, followed by a longer-lasting increase, in systemic vascular resistance. In contrast to ET-1, big endothelin-1 (bET-1) produces monophasic increases in systemic vascular resistance and arterial blood pressure. This is somewhat surprising, because bET-1 activity is reportedly due to ET-1, bET-1 being converted to ET-1 by a putative converting enzyme. In this study we tested two hypotheses that could explain the singular effect of bET-1 on the arterial vasculature: that bET-1 vasoconstriction, mediated by ETA receptors at the level of the smooth muscle, masks the release of endothelial derived vasodilators, and/or that the endothelium develops tachyphylaxis owing to prolonged activation of endothelial ETB receptors. In anesthetized rats, blockade of the vasoconstrictor activity of bET-1 with BQ-123, an ETA-receptor antagonist, did not reveal a masked bET-1 vasodilator component in the rat hindquarter. Furthermore, in the presence of bET-1 (after 3.0 nmol/kg bET-1 i.v.), low doses of ET-1 (0.03-0.3 nmol/kg) produced dose-dependent hindquarter vasodilation, indicating activation of endothelial ETB and therefore no tachyphylaxis. Collectively, these experiments suggest that i.v. administration of bET-1 results in little or no activation of endothelial ETB receptors and therefore lacks a vasodilator response.