Characterization of endothelin-1 receptor subtypes in isolated human cardiomyocytes.

On cardiac membranes and isolated cardiomyocytes from the human heart, cell-type distribution and functional activities of endothelin-1 (ET-1) receptor subtypes were investigated by using binding methods and messenger RNA (mRNA) in situ hybridization. The ET-receptor antagonist BMS-182874 selectively and competitively inhibits ET(A) receptors both on isolated myocytes and ventricular membranes with approximately 1,300 times greater affinity for ET(A) than ET(B) subtypes. The [125I]-ET-1 specific binding revealed 42.851+/-2,546 receptors/myocyte with a prevalent proportion of ET(A)-receptor subtypes on both myocytes (84+/-2%) and ventricular membranes (66+/-3%). In situ hybridization studies revealed that mRNA for ET(A) receptors was expressed on both myocytes and nonmyocyte cells, whereas mRNA for ET(B) receptors was almost exclusively expressed on fibroblasts and endothelial cells. Specific binding of [125I]-ET-1 to both myocytes and ventricular membranes in the presence of specific ET(A) (BMS-182874) and ET(B) (BQ-788)-receptor antagonists showed a displacement of [125I]-ET-1 by unlabeled ET-1, which were significantly faster from ET(B) than from ET(A). This suggests a clearance function of ventricular ET(B) receptors.     

Drug Discovery for Overcoming Chronic Kidney Disease (CKD): The Endothelin ETB Receptor / Nitric Oxide System Functions as a Protective Factor in CKD

Accelerated cardiovascular disease (CVD) is a frequent complication of renal disease. Chronic kidney disease (CKD) develops hypertension and dyslipidemia, which in turn can contribute to the progression of renal failure. There is general agreement that endothelin-1 (ET-1), which acts through the two subtypes of receptor ET_A and ET_B, plays important physiological roles in the regulation of normal cardiovascular function and that excessive ET-1 production is linked to CVD and CKD. Although selective ET_A or nonselective ET_A/ET_B receptor antagonisms have been recognized as a potential strategy for treatment of several cardiovascular disease, it remains unclear which of the antagonisms is suitable for the individuals with CKD because upregulation of the nitric oxide (NO) system via ET_B receptor is responsible for renal function such as natriuresis, diuresis, and glomerular hemodynamics. Our findings clearly indicate that the blockade of ET receptors, in particular ET_A-receptor antagonism, not only produces a potential renoprotective effect in CKD but also reduces the risk of CVD. In contrast, pharmacological blockade or genetic deficiency of ET_B receptor seems to aggravate CKD and CVD in several experimental models of rats. Moreover, preliminary evidence in patients with CKD also suggests that both selective ET_A- and nonselective ET_A/ET_B-receptor blockade decreases blood pressure but that selective ET_A blockade has additional desirable effects on renal hemodynamics. Thus, at least in CKD, these findings support the notion that ET_B receptor– mediated actions produce a renoprotective effect and that nonselective ET_A/ET_B-receptors blockade seem to offer no advantage over selective ET_A antagonism, and if anything may potentially reduce the benefits.     

Enhanced endothelium-dependent vasodilatation by dual endothelin receptor blockade in individuals with insulin resistance

Insulin resistance is associated with endothelial dysfunction and increased production of the pro-inflammatory vasoconstrictor peptide endothelin-1 (ET-1). The aim of this study was to test the hypothesis that blockade of ET receptors results in enhanced endothelium-dependent vasodilatation (EDV) in individuals with insulin resistance. Twelve individuals with insulin resistance without any history of diabetes or cardiovascular disease and 8 age-matched controls with high insulin sensitivity, as determined by hyperinsulinemic-euglycemic clamp, were investigated on 2 separate occasions using forearm venous occlusion plethysmography. Endothelium-dependent and endothelium-independent vasodilatation was determined before and after selective ET(A) and dual ET(A)/ET(B) receptor blockade. A 60 minute intraarterial infusion of the ET(A) receptor antagonist BQ123 (10 nmol/min) combined with the ET(B) receptor antagonist BQ788 (5 nmol/min) evoked a significant increase in acetylcholine-mediated EDV (P < 0.01) in individuals with insulin resistance. The endothelium-independent vasodilator response to nitroprusside was not changed by dual ET(A)/ET(B) receptor blockade. Dual ET(A)/ET(B) receptor blockade did not affect the response to acetylcholine or nitroprusside in the insulin-sensitive group. Selective ET(A) receptor blockade did not evoke any changes in endothelium-dependent or endothelium-independent vasodilatation in either group. This study demonstrates that dual ET(A)/ET(B) receptor blockade, but not selective ET(A) blockade, enhances EDV in subjects with insulin resistance, suggesting that ET-1 is involved in the regulation of endothelial function in individuals with insulin resistance.     

Distribution of endothelin receptors in saphenous veins of African Americans: implications of racial differences.

The relative density of endothelin-receptor subtypes A and B (ET(A) and ET(B), respectively) on endothelial and smooth muscle cells is the major determinant of the contractile response to endothelin-1 (ET-1). To investigate the effects of race on the distribution of ET receptors, the endothelin-receptor subtypes on membrane fractions prepared from saphenous veins obtained from African-American patients undergoing coronary bypass surgery were analyzed. Similar studies were repeated with endothelium-denuded samples to study the role of endothelium- and smooth muscle-derived ET(B) receptors. Competitive-binding experiments on membrane fractions by using [125I]-ET-1 and unlabeled ligands, ET-1, ET-3, sarafotoxin-6-c, and BQ-123 yielded two classes of binding sites on endothelium-intact vessels from both female and male subjects. In women, the maximal binding capacities were 91+/-6 and 67+/-13 fmol/mg protein for ET(A) and ET(B) receptors, respectively; the corresponding values in men were 178+/-19 and 127+/-13 fmol/mg protein. Similar experiments performed with endothelium-denuded saphenous veins indicated the presence of both receptor subtypes on vascular smooth muscle, in contrast to our earlier report on the presence of only ET(A) receptors on vessels obtained from white Americans. Our findings demonstrate that the ratio and the density of ET receptors are different in black and white Americans.     

Endothelin receptor antagonists: structures, synthesis, selectivity and therapeutic applications

Endothelin (ET) was discovered in 1988 and is the most potent vasoconstrictive peptide known to date. It exists in three isoforms (ET-1 to ET-3) and acts on two endothelin receptor subtypes, the endothelin-A (ET(A))-receptor and the endothelin-B (ET(B))-receptor. Endothelin receptor antagonists are novel therapeutics in clinical development for different cardiovascular, cerebrovascular, and renal diseases. Several different structural classes of endothelin receptor antagonists have been discovered within the last decade, starting from peptidic- and peptidomimetic structures to small organic molecules suitable as therapeutics for oral administration. Focussing on the small organic molecules, the different structural classes of ET-receptor antagonists are described with respect to synthesis, structure-activity-relationships, receptor-subtype-selectivity profile, and where possible, intended therapeutic indications.     

Differential effects of the mixed ETA/ETB-receptor antagonist bosentan on endothelin-induced bronchoconstriction, vasoconstriction and prostacyclin release

Endothelins are a family of potent endogenous mediators that have been implicated in a number of airway and other diseases. Recently, the non-peptide mixed ET(A)/ET(B) endothelin receptor antagonist bosentan has been successfully tested in the treatment of cardiovascular diseases. It was the aim of the present study to characterize the effects of bosentan on the pulmonary actions of endothelin- (ET-1), endothelin-3 (ET-3) and the ET(B)-receptor agonist IRL1620 in the isolated perfused and ventilated rat lung (IPL) and in precision-cut lung slices (PCLS). In the IPL, bosentan completely prevented the IRL1620-induced vasoconstriction (IC50 3 microM). The inhibition by bosentan of ET-1-elicited vasoconstriction showed a biphasic course, reflecting the inhibition of ET(A)-and ET(B)-mediated vasoconstriction (IC50 0.2 microM and 19 microM, respectively). In addition, bosentan prevented the ET-1- (IC50 6 microM) and IRL1620-induced (IC50 3 microM) prostacyclin release. Bosentan also completely prevented the bronchoconstriction induced by IRL1620 in the IPL (IC50 20 microM) and in PCLS (IC50 13 microM). In PCLS, the pD2-values were ET-1 7.20+/-0.23, ET-3 7.51+/-0.27 and IRL1620 7.33+/-0.29. Bosentan at 100 microM caused a rightward shift of the concentration-response curve of ET-1, ET-3 and IRL1620 by a factor of 5, 46 and 64, respectively. In all cases the slope of the Schild regression was lower than unity, disregarding a simple interaction of bosentan with one receptor. With respect to ET-1-induced bronchoconstriction, in the IPL bosentan in concentrations of up to 10 microM aggravated ET-1-induced bronchoconstriction probably due to the blockade of bronchodilatory ET(A)-receptors (IC50 0.3 microM) and even at 100 microM showed only very little protection from ET- -induced bronchoconstriction in the IPL and in the PCLS. The similar IC50-values for ET-1-induced vasoconstriction and bronchodilation suggest that only one type of ET(A)-receptor is involved. The differing IC50-values between IRL1620-induced bronchoconstriction and prostacyclin release, the slope of the Schild regression and the failure of bosentan to prevent the ET-1-induced bronchoconstriction suggest a complex interaction between the known ET-receptors or the existence of unknown ET(B)-receptor subtypes.     

Endothelin-1 and endothelin receptor antagonists as potential cardiovascular therapeutic agents.

Endothelin (ET)-1 is an endothelium-derived peptide with potent vasoconstrictor and proliferative properties. The ET system is activated in several cardiovascular disease states associated with functional and structural vascular changes, including hypertension and heart failure. The two ET receptor subtypes are known as ET(A)R and ET(B)R. The former is located mainly on vascular smooth muscle cells and is responsible for mediating vasoconstriction and proliferation. The latter is present predominantly on endothelial cells and mediates vasorelaxation as well as ET-1 clearance. Activation of smooth muscle ET(B)R causes vasoconstriction. Selective ET(A)R antagonists as well as nonselective ET(A)R-ET(B)R antagonists have been developed. Studies with animal models and early-phase clinical trials provided strong evidence that these agents are effective in the treatment of heart failure, essential hypertension, pulmonary hypertension, and atherosclerosis. However, the complexity of biologic effects mediated by two different receptor subtypes complicates therapy with selective versus nonselective ET receptor antagonists. In addition to subtype selectivity and potency, changes in receptor subtype distribution under different pathologic conditions and different patient populations will play a crucial role in the evaluation of these potentially therapeutic drugs.     

Receptor subtypes mediating the inotropic effects and Ca(2+) signaling induced by endothelin-1 through crosstalk with norepinephrine in canine ventricular myocardium

In canine ventricular myocardium, endothelin-1 (ET-1) alone induced only a weak transient negative inotropic effect (NIE). However, ET-1 induced a marked sustained positive inotropic effect (PIE) subsequent to a transient NIE in the presence of norepinephrine (NE) at low concentrations (0.1 - 1 nM) and elicited a pronounced sustained NIE in the presence of NE at high concentrations (around 100 nM). Thus, the extent of beta-adrenoceptor stimulation induced by NE played a crucial role in determining the characteristics of the inotropic effects of ET-1. The characteristics of ET receptor subtypes involved in contractile regulation and Ca(2+) signaling induced by ET-1 were determined. The ET-1-induced transient NIE and decrease in Ca(2+) transients were abolished by the selective ET(A)-receptor antagonist FR319317, but not by the selective ET(B)-receptor antagonist BQ-788. The sustained PIE and the increase in Ca(2+) transients induced by ET-1 were abolished by FR319317, but not inhibited by BQ-788. In contrast, the sustained NIE of ET-1 was abolished by the non-selective ET antagonist TAK-044, markedly attenuated by FR319317, and partially inhibited by BQ-788. ET-1 alone elicited a PIE in the presence of BQ-788, which indicates that the activation of ET(B)-receptors counteracts the development of the PIE of ET-1. The current findings indicate that both ET(A) and ET(B) receptors are involved in the regulation of Ca(2+) signaling and contractility in canine ventricular myocardium.     

Evidence for vasoconstriction mediated by the endothelin-B receptor in domestic swine

Endothelin-1 (ET-1), a potent vasoactive and mitogenic peptide, has been implicated in a number of cardiovascular diseases, including congestive heart failure, neointimal hyperplasia associated with restenosis, and hypertension. The vasoconstriction induced by ET-1 is thought to be mediated mainly by its action on ET(A) receptors on vascular smooth muscle cells. Recent studies have indicated that vasoconstriction also may be mediated by stimulation of an ET(B)-receptor subtype. Increased use of the pig as a cardiovascular model prompted us to examine the receptor profile in this species using ABT-627, a potent, nonpeptide antagonist of the ET(A) receptor. The precursor to ET-1, big ET-1 (0.02 nmol/kg/min), was infused intravenously in domestic swine, resulting in a sustained increase in mean blood pressure of 38 +/- 3 mm Hg. After stabilization of the pressor response, ABT-627 (0.1-10 microg/kg/min) or vehicle was infused for 30 min. Whereas vehicle infusion had no appreciable effect, a dose-related reversal of the pressor response to big ET-1 (11-100%) was observed by the end of the ABT-627 infusion. Blood samples were assayed for plasma concentrations of ABT-627; peak levels ranged from 9 +/- 2 to 937 +/- 168 ng/ml. In a separate group of pigs, the highest dose of ABT-627 produced only a modest reversal of the hypertensive response to an infusion of angiotensin II (300 ng/kg/min). Additional results indicate that the vasoconstrictor effects produced by sarafotoxin 6C (0.03 and 0.3 nmol/kg), an agonist of the ET(B) receptor, are not blocked by treatment with ABT-627 (10 microg/kg/min). However, complete blockade of the S6C response could be achieved using the ET(B) antagonist, A-192621 (0.33 mg/kg/min). Our results define the dose-response relation for the ET(A)-receptor antagonist ABT-627 in the vasculature of the domestic pig and suggest the presence of an ET(B)-receptor subtype that mediates vasoconstriction in this species.     

Enhanced expression of contractile endothelin ET(B) receptors in rat coronary artery after organ culture

Endothelin-1 is a potent vasoconstrictor mediating its effects via two receptor subtypes, the endothelin type A (ET(A)) preferentially situated on smooth muscle cells, mediating vasoconstriction and endothelin type B (ET(B)) mainly located on endothelial cells, mediating vasodilatation. In cardiovascular disease and in organ culture in vitro, endothelin ET(B) receptors are up-regulated on smooth muscle cells. The objectives of the present study were to characterise the endothelin receptor-induced vasoconstriction and quantify the endothelin receptor mRNA levels and immunoreactivity in fresh and cultured rat coronary arteries. We demonstrate that endothelin-1 induces strong and equal concentration-dependent contractions in fresh and cultured segments from the left anterior descending coronary artery. Sarafotoxin 6c, an endothelin ET(B) receptor agonist, had negligible effect in fresh arteries but produced significant vasoconstriction after organ culture. The endothelin ET(B) receptor mRNA level and the receptor protein immunoreactivity were increased, whereas the level of endothelin ET(A) receptor mRNA was down-regulated but not its receptor protein immunoreactivity after organ culture. Pharmacological inhibition of endothelium-derived dilatory mediators did not influence endothelin ET(A) or ET(B) receptor-mediated vasoconstriction in fresh segments. In cultured arteries, inhibition of endothelial vasodilators potentiated the effect of sarafotoxin 6c. In conclusion, endothelin ET(B) receptor stimulation in cultured coronary arteries elicits vasoconstriction. This is likely not related to endothelial dysfunction with putative loss of its vasodilator components, but rather explained by the up-regulation of contractile endothelin ET(B) receptors on smooth muscle cells.     

Pharmacological characterization of endothelin receptors-mediated contraction in the mouse isolated proximal and distal colon

The study investigated the role of endothelin (ET) and the ET receptor subtypes ET(A) and ET(B) in mediating longitudinal contraction in the mouse proximal and distal colon. Cumulative concentration-response curves to a range of ET agonists (ET-1, ET-2, ET-3, (Ala(1,3,11,13)) ET and IRL 1620) were established by administering concentrations ranging from 0.01 nM to 0.3 microM. Concentration-response curves to ET-1, which exhibits a high affinity for both ET(A) and ET(B) receptor subtypes, were also established in the presence of the ET(A) antagonist BMS 182874 and the ET(B) antagonist IRL1038. The addition of the selective ET(A) receptor antagonist BMS 182874 caused a rightward shift of the concentration-response curve to ET-1 in both sections of the colon. The ET(B) receptor antagonist IRL1038 (0.3-1 microM) did not significantly effect the response to ET-1 in the proximal colon but caused a significant decrease in response towards higher concentrations ranges (>or=3 nM) in the distal colon. A comparison of the concentration-response curves to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1>or=ET-2>ET-3 in the proximal colon and ET-1>or=ET-2>or=ET-3 in the distal colon. The selective ET(B) receptor agonists, (Ala(1,3,11,13)) ET and IRL 1620 did not produce any response in the proximal sections of the colon but produced a smaller contraction in the distal segments. The data indicate that ET can contract the proximal tissues of the mouse colon predominantly via ET(A) receptors and in the distal tissues via ET(A) and ET(B) receptors.     

Involvement of endothelin and ET(A) endothelin receptor in mechanical allodynia in mice given orthotopic melanoma inoculation

We investigated whether endothelin (ET) would be involved in skin cancer pain in mice. Orthotopic inoculation of B16-BL6 melanoma cells into the plantar region of the hind paw produced marked mechanical allodynia in C57BL/6 mice. Intraplantar injections of the ET(A)-receptor antagonist BQ-123 (0.3 - 3 nmol/site), but not the ET(B)-receptor antagonist BQ-788 (1 and 3 nmol/site), inhibited mechanical allodynia in mice with grown melanoma. In naive mice, an intraplantar injection of tumor extract (1 and 3 mg/site), which was prepared from the grown melanoma in the paw, produced mechanical allodynia, which was inhibited by BQ-123 and BQ-788 at doses of 3 and 10 nmol/site. An intraplantar injection of ET-1 (1 and 10 pmol/site) elicited licking behavior, which was increased in the melanoma-bearing hind paw. BQ-123 (3 and 10 nmol/site) inhibited licking induced by ET-1 (10 pmol/site). The level of mRNA of ET(A), but not ET(B), receptor, was significantly increased in the dorsal root ganglia on the inoculated side. Cultured B16-BL6 cells contained ET, and the melanoma mass increased the concentration of ET as it grew bigger. These results suggest that ET-1 and ET(A) receptor are at least partly involved in the induction of pain induced by melanoma cell inoculation.     

Evidence that ET-1, but not ET-3 and S6b, ET(A)-receptor mediated contractions in isolated rat mesenteric arteries are modulated by co-activation of ET(B) receptors.

The effects of agonists with endothelin (ET) ET(A)-receptor activity have been analysed in relation to their interaction with ET(B) receptors in rat mesenteric arteries. ET-1, sarafotoxin 6b (S6b) and ET-3 induced large, slow-onset and sustained contractions whereas S6c induced weak transient contractions. However, following pre-contraction with U46619 and subsequent relaxation with forskolin, the effect of S6c was amplified, indicating a potential for powerful ET(B)-receptor mediated contraction. The selective ET(A)-receptor antagonist, FR139317, produced parallel rightward shifts of ET-1, S6b and ET-3 concentration-effect curves indicating that the contractions were mediated by ET(A) receptors. However, the corresponding FR139317 pK(B) values were significantly different between the agonists. As expected FR139317 had no effect on S6c responses. Pre-treatment with S6c to desensitize ET(B) receptors, increased ET-1 potency and the pK(B) value for FR139317. In contrast, neither the potency of S6b and ET-3 nor the pK(B) values for FR139317 estimated using these agonists were affected by ET(B)-receptor desensitization. Segments pre-contracted with submaximal concentrations of S6b and ET-3, but not ET-1, rapidly relaxed following wash-out or FR139317 administration. The results indicate that the small contractile response to selective ET(B) receptor activation, barely detectable under standard bioassay conditions, is greatly amplified when adenylate cyclase activity is elevated. Moreover, the response to ET(A) receptor activation by ET-1, but not ET-3 and S6b, is significantly modified by co-activation of ET(B) receptors. This interaction has a significant effect on the apparent affinity of ET(A)-receptor selective antagonists when ET-1 is used as agonist and decreases the potency of ET-1.     

BQ-788, a selective endothelin ET(B) receptor antagonist

We describe characteristics of a selective endothelin (ET) ET(B) receptor antagonist, BQ-788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine], which is widely used to demonstrate the role of endogenous or exogenous ETs in vitro and in vivo. In vitro, BQ-788 potently and competitively inhibited (125)I-labeled ET-1 binding to ET(B) receptors in human Girrardi heart cells (hGH) with an IC(50) of 1.2 nM, but only poorly inhibited the binding to ET A receptors in human neuroblastoma cell line SK-N-MC cells (IC(50), 1300 nM). In isolated rabbit pulmonary arteries, BQ-788 showed no agonistic activity up to 10 microM and competitively inhibited the vasoconstriction induced by an ET(B)-selective agonist (pA(2), 8.4). BQ-788 also inhibited several bioactivities of ET-1, such as bronchoconstriction, cell proliferation, and clearance of perfused ET-1. Thus, it is confirmed that BQ-788 is a potent, selective ET(B) receptor antagonist. In vivo, in conscious rats, BQ-788, 3 mg/kg/h, i.v., completely inhibited a pharmacological dose of ET-1- or sarafotoxin6c (S6c) (0.5 nmol/kg, i.v.)-induced ET(B) receptor-mediated depressor, but not pressor responses. Furthermore, BQ-788 markedly increased the plasma concentration of ET-1, which is considered an index of potential ET(B) receptor blockade in vivo. In Dahl salt-sensitive hypertensive (DS) rats, BQ-788, 3 mg/kg/h, i.v., increased blood pressure by about 20 mm Hg. It is reported that BQ-788 also inhibited ET-1-induced bronchoconstriction, tumor growth and lipopolysaccharide-induced organ failure. These data suggest that BQ-788 is a good tool for demonstrating the role of ET-1 and ET(B) receptor subtypes in physiological and/or pathophysiological conditions.     

Role of endothelin in the cardiovascular system

The endothelin (ET) system consists of three peptide ligands (ET-1, ET-2 and ET-3) and two G-protein-coupled receptors, ET_A and ET_B. In the cardiovascular system, ETs, particularly ET-1, are expressed in smooth muscle cells, cardiomyocytes, fibroblasts, and notably in vascular endothelial cells. Intense research over the last 10 years has changed the original view of ET-1 as mainly a vasoconstrictor regulating blood pressure, into a biological factor regulating processes such as vascular remodeling, angiogenesis or extracellular matrix synthesis. The advent of specific (and type-selective) ET receptor antagonists has greatly fostered our knowledge of the biological function of ET-1, and has offered a potential therapeutic approach for numerous diseases including hypertension, atherosclerosis or fibrosis. In this article, we review the regulation of the expression of vascular ET-1, as well as the contribution of ET-1 to endothelial, smooth muscle and fibroblast cell function, with particular interest in the role of ET-1 in the development of cardiovascular diseases.     

Endothelin receptor-mediated vasodilatation: effects of organ culture

Culture of intact arteries is a frequently employed experimental model for investigating the mechanisms governing the regulation of vascular endothelin receptors. Endothelin type A (ET(A)) and type B (ET(B)) receptors on vascular smooth muscle cells are up-regulated in organ culture and the enhanced vasoconstriction mimics the changes that occur in cardiovascular disease. The effect of organ culture on endothelial dilatory endothelin ET(B) receptors is not known. We hypothesize that organ culture decreases the endothelin receptor-mediated dilatation and that this is one possible mechanism by which the effects of the endothelin in blood vessels are altered during culture. Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology and immunofluorescence. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ET(A) and ET(B) receptor effects, and the antagonists, Nomega-nitro-l-arginine (l-NOARG) for nitric oxide (NO), indomethacin for prostaglandins and charybdotoxin in combination with apamin for endothelium-derived hyperpolarizing factor (EDHF), were used to study the endothelium-derived dilatory mediators. Organ culture induced up-regulation of the sarafotoxin 6c (ET(B) receptor agonist) and endothelin-1 (ET(A) receptor agonist) elicited vasoconstriction. The sarafotoxin 6c contraction was stronger after endothelium denudation, suggesting endothelium-dependent dilatation. The endothelin-1 contraction was not affected by endothelium denudation. The increase in sarafotoxin 6c contraction after removal of the endothelium was more pronounced before than after organ culture, suggesting down-regulated endothelial endothelin ET(B) receptors. Also, the immunofluorescence staining intensities for endothelial endothelin ET(B) receptors were higher before than after organ culture. Pre-incubation with inhibitors for dilatory mediators suggested that both NO and EDHF play a vasodilatory role, while prostaglandins are not involved. In conclusion, endothelial endothelin ET(B) receptors induce NO and EDHF mediated vasodilatation in porcine coronary arteries. In organ culture, endothelial endothelin ET(B) receptors are down-regulated, mimicking the changes that occur in cardiovascular disease. Down-regulation of endothelial endothelin ET(B) receptors may in part explain the increased endothelin ET(B) receptor-mediated vasoconstriction frequently studied in organ culture.     

Pathophysiological role of endothelin-1 in the vascular remodeling process

Endothelin (ET)-1, which had been discovered as the most potent vasoconstrictive peptide, plays active roles in the various biological responses. However, it is controversial how is ET-1 involved in the vascular remodeling process. Therefore, present experiments were performed to investigate the role of ET-1 for the initiation/progression of intimal hyperplasia. Intimal hyperplasia was caused with the time to peak of 4 to 6 weeks after endothelial removal of the rabbit carotid artery and accompanied by increased ET-1 content as well as ET receptor density within the hyperplastic vessel wall. ET-1 receptors could be classified into three subtypes of ETA, ETB and tentative nonETA/nonETB. TUNEL- and Ki-67-positive cells were detectable with the time to peak of 1 to 2 weeks, whereas all positive cells disappeared within 6 weeks. ATZ1993 as a mixed type antagonist for ET-A, ET-B and nonETA/nonETB receptor subtypes inhibited the intimal hyperplasia, of which inhibition was accompanied by increased TUNEL- and p53-positive cells and decreased Bcl-2-positive cells. ET-1 accelerated the [3H]-thymidine incorporation by cultured vascular smooth muscle cells and, on the other hand, reduced TUNEL-positive cells, which was caused by the serum deprivation. The reduction of TUNEL-positive cells with ET-1 was blocked by ATZ1993 or BQ123 as an antagonist for ETA receptor, but unaffected by BQ788 as an antagonist for ETB receptor. These results strongly suggest that ET-1 plays crucial roles as a mitogen and an inhibitory factor of apoptosis in the vascular remodeling process after endothelial removal.     

Differential modulation of endothelin ligand-induced contraction in isolated tracheae from endothelin B (ET(B)) receptor knockout mice

The role of endothelin B (ET(B)) receptors in mediating ET ligand-induced contractions in mouse trachea was examined in ET(B) receptor knockout animals. Autoradiographic binding studies, using [(125)I]-ET-1, confirmed the presence of ET(A) receptors in tracheal and bronchial airway smooth muscle from wild-type (+/+) and homozygous recessive (-/-) ET(B) receptor knockout mice. In contrast, ET(B) receptors were not detected in airway tissues from (-/-) mice. In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET-1>ET-3; Stx S6c had a lower efficacy than ET-1 or ET-3. In tissues from (-/-) mice there was no response to Stx S6c (up to 0.1 microM), whereas the maximum responses and potencies of ET-1 and ET-3 were similar to those in (+/+) tracheae. ET-3 concentration-response curve was biphasic in (+/+) tissues (via ET(A) and ET(B) receptor activation), and monophasic in (-/-) preparations (via stimulation of only ET(A) receptors). In (+/+) preparations SB 234551 (1 nM), an ET(A) receptor-selective antagonist, inhibited the secondary phase, but not the first phase, of the ET-3 concentration-response curve, whereas A192621 (100 nM), an ET(B) receptor-selective antagonist, had the opposite effect. In (-/-) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET-3 concentration-response curves. The results confirm the significant influence of both ET(A) and ET(B) receptors in mediating ET-1-induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ET(B) receptors. In this preparation ET-3 is not an ET(B) receptor-selective ligand, producing contractions via activation of both ET(A) and ET(B) receptors.