SB 209670, a rationally designed potent nonpeptide endothelin receptor antagonist.

An extremely potent and highly specific non-peptide, subnanomolar endothelin (ET) receptor antagonist, SB 209670, has been synthesized and characterized. SB 209670, which was rationally designed using conformational models of ET-1, selectively inhibits binding of 125I-labeled ET-1 to cloned human ET receptor subtypes ETA and ETB (Ki = 0.2 and 18 nM, respectively). SB 209670 produces concentration-dependent inhibition of ET-1-mediated vasoconstriction in isolated vascular tissues and in vivo following either intravenous or intraduodenal administration. SB 209670 produces a dose-dependent reduction in blood pressure in hypertensive rats, protects from ischemia-induced neuronal degeneration in a gerbil stroke model, and attenuates neointima formation following rat carotid artery balloon angioplasty. SB 209670 will be useful in characterizing and classifying the physiological and pathophysiological effects of ET.     

Receptor- and non-receptor-mediated clearance of big-endothelin and endothelin-1: differential effects of acute and chronic ETA receptor blockade

AIMS: The aims of this study were to define and characterize the different mechanisms and sites of clearance of plasma endothelin-1 (ET-1) and big endothelin-1 (BigET-1) and evaluate possible effects of ETA versus combined ETA and ETB receptor blockade or endothelin converting enzyme (ECE) inhibition. METHODS: Time courses and sites of clearance were evaluated in Wistar-Kyoto rats after bolus injection of radiolabelled peptides into the carotid artery before or after treatment with LU1 35252 (ETA) and bosentan (ETA and ETB) as receptor antagonists or the ECE inhibitor phosphoramidon. RESULTS: The study shows that differential clearance of 125I-ET-1 and 125I-BigET-1 is mediated by distinct tissue-specific, receptor- and non-receptor-mediated mechanisms. Low levels of plasma ET-1 are rapidly cleared, mainly in the pulmonary circulation, through a low-capacity saturable ETB receptor-linked mechanism. In contrast, BigET-1 clearance is markedly slower, confined largely to liver and kidneys, is essentially non-receptor-mediated and is independent of converting enzyme activity. Acute inhibition of both ETA and ETB receptors with bosentan dramatically prolonged 125I-ET-1 plasma half-life and shifted tissue uptake from lung to liver and kidneys. Pulmonary clearance of 125I-ET-1 was decreased by chronic but not acute treatment with the specific ETA receptor antagonist LU135252. In contrast, 125I-Big-ET-1 clearance and tissue uptake were essentially unchanged by all treatments. CONCLUSIONS: Plasma levels and clearance studies on ET-1 and BigET-1 may provide differential information regarding pathological changes in their separate uptake mechanisms. Such data could have diagnostic or prognostic value in pulmonary, hepatic and renal pathophysiology or future therapeutic monitoring of treatment efficacy following administration of selective receptor antagonists.     

Expression of endothelin-converting enzyme, endothelin-1 and endothelin receptors at the site of percutaneous coronary intervention in humans

OBJECTIVE: The repair process at the site of injury after percutaneous coronary intervention (PCI) is dominated by neointimal formation composed mainly of smooth muscle cells (SMC). Endothelin-1 (ET-1) is a powerful vasoconstrictor and SMC mitogen. Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin processing. The effects of ET-1 are mediated by binding to endothelin type A (ETA) and endothelin type B (ETB) receptors. The ligand/receptor/ligand-producing system (ET system) could be involved in the pathogenesis of neointimal formation in humans. METHODS: Fifteen post-PCI sites obtained at autopsy and eight atherectomy specimens obtained from restenotic sites were investigated using immunohistochemical single and double staining techniques. Frozen sections were stained with antibodies against ECE, ET-1, ETA and ETB receptors, SMC, macrophages and endothelial cells. RESULTS: At the early stage, less than 3 months after PCI, neointimal SMC were positive for ECE, ET-1, ETA and ETB receptors. The expression of ECE, ET-1, ETA and ETB receptors in these neointimal SMC decreased markedly from 6 months onwards. The ECE, ET-1, ETA and ETB receptor-positive cell areas were significantly (P < 0.005) greater in the first 3 months after PCI compared with 6 months or more after PCI. Atherectomy specimens also showed similar positivity. CONCLUSIONS: These observations strongly suggest that the expression of ECE, ET-1, ETA and ETB receptors is enhanced in neointimal SMC at early stages after PCI injury in human coronary arteries. The increased expression of the ET system may contribute to SMC proliferation/migration and vasoconstriction in human post-PCI coronary lesions.     

Improvement of renal dysfunction in rats with chronic heart failure after myocardial infarction by treatment with the endothelin A receptor antagonist, LU 135252

OBJECTIVE: To investigate the role of an activated endothelin system in the renal dysfunction observed in chronic heart failure after myocardial infarction. METHODS: In rats with heart failure after myocardial infarction and in sham-operated animals (Sham), we investigated the effect on renal function of long-term oral treatment with the selective endothelin A (ETA) receptor antagonist, LU 135252 (30 mg/kg per day; groups MI/LU and Sham/LU) or placebo (groups MI/P, Sham/P). Only animals with extensive myocardial infarction (at least 46% of the left ventricle) were included in the study. Infarct size was matched between groups MI/P and MI/LU. Endogenous creatinine clearance, fractional sodium excretion, and plasma and urinary concentrations of endothelin were determined 12 weeks after myocardial infarction. RESULTS: Endogenous creatinine clearance was significantly lower in group MI/P than in group Sham/P (MI/P: 0.64 +/- 0.05, Sham/P: 0.81 +/- 0.04 ml/min per 100 g body weight; P= 0.01 (means +/- SEM)). Treatment with LU 135252 completely prevented the decline in creatinine clearance in rats with chronic myocardial infarction (MI/LU: 0.98 +/- 0.21; Sham/LU: 0.83 +/- 0.10). Fractional sodium and protein excretion did not differ among the four groups. Group MI/P had a marked increase in plasma endothelin concentrations, which was not affected by treatment with LU 135252. Urinary endothelin excretion was significantly lower in group MI/P than in group Sham/P. In the treatment groups, no difference could be observed between animals that had suffered myocardial infarction and the sham-operated group, although LU 135252 markedly increased the urinary excretion of endothelin. CONCLUSION: Our data demonstrate a restoration of impaired renal function in chronic ischaemic heart failure by treatment with the selective ETA receptor antagonist, LU 135252. These results offer a promising therapeutic option for the treatment of renal insufficiency in patients with chronic heart failure.     

Endothelins and ischemic heart disease

Endothelins (ET) comprise a group of substances which are produced and have a regulatory function in different systems of the organism. The main cardiovascular ET is ET-1 which is so far the most potent vasoconstrictor substance. ET is important in the early stages of atherosclerosis as it is a strong chemical attractant of monocytes and macrophages and causes endothelial and vasomotor dysfunction. In later stages of atherosclerosis it can affect the firmness and integrity of the fibrous part of the atherosclerotic plaque. During myocardial infarction the ET level rises and this leads to vasoconstriction and reduction of the fibrillation threshold. In coronary angioplasty (PTCA) the ET-1 level rises depending on the applied mechanical stress. ET can participate in the formation of the neointima and the development of restenosis. ET increases also immediately after an aortocoronary bypass (CABG) and after reperfusion. Higher ET levels were found in patients with a positive ECG and echocardiographic loading test. In the latter after CABG normalization of ET was found. Raised ET levels were recorded also in patients where a coronary vasospasm can be provoked, in Prinzmetal's angina pectoris, coronary syndrome X, atrial tachystimulation. In unstable angina pectoris big-ET is elevated, the increase of the ET-1 level is not unequivocal. Stable angina pectoris does not affect ET-1. In the treatment of atherosclerosis for the selective ETA blocker reduction of the number and size of macrophage and foam cells was proved. In acute myocardial infarction a favourable effect for the non-selective ETA/ETB blocker and for the selective ETA blocker was found. In the treatment of restenosis after PTCA blockers of selective ETB receptors and inhibitors of endothelin converting enzyme seem hopeful. Th non-selective ETA/ETB blocker bosentan is in the stage of clinical tests and seems to be safe and perspective.     

In-vivo interaction of nitric oxide and endothelin

OBJECTIVE AND METHODS: Endothelin-1 (ET-1) was initially characterized as a potent vasoconstrictor. However, the expected role of ET-1 as a major blood pressure controlling peptide could not be clearly established. Moreover, ET-1 transgenic mice are not hypertensive. We assume that counter-regulating mechanisms such as the nitric oxide (NO) system or an altered expression of endothelin receptors might cause this finding. RESULTS: An intravenous (i.v.) bolus injection of N(omega)-nitro-L-arginine methyl ester (L-NAME) resulted in a significantly higher blood pressure increase in ET-1 transgenic mice, as compared to non-transgenic littermates. On the other hand, blood pressure increased similarly after an i.v. injection of ET-1 in ET-1 transgenic mice and non-transgenic littermates. Pretreatment with dexamethasone abolished the higher blood pressure increase after L-NAME in ET-1 transgenic mice. Urinary excretion of NO metabolites was elevated in ET-1 transgenic mice and decreased significantly after dexamethasone treatment. Immunohistochemistry revealed that the inducible NO synthase (iNOS) was highly expressed in intrarenal arteries in these mice. Dexamethasone pretreatment abolished vascular iNOS expression. No vascular iNOS expression was detectable in non-transgenic littermates. Furthermore, immunohistochemistry revealed that ET-1 transgenic mice are characterized by an increased tissue density of CD4-positive lymphocytes and macrophages. Analysis of endothelin receptor expression and function revealed that the endothelin subtype A (ETA) receptor was not differently expressed in ET-1 transgenic mice as compared to age-matched littermates. The blood pressure response to an ETA receptor antagonist was likewise similar in ET-1 transgenic mice and age-matched littermates. The endothelin subtype B (ETB) receptor density was decreased in ET-1 transgenic mice. Treatment with an ETB receptor antagonist led to a non-significant slightly higher blood pressure increase in ET-1 transgenic mice as compared to controls. CONCLUSION: The endothelin receptor expression pattern and the blood pressure responses to ETA and ETB receptor antagonists could not explain the lack of hypertension in ET-1 transgenic mice. Overexpression of the human ET-1 gene causes chronic kidney inflammation with an induction of vascular iNOS expression. The induction of iNOS expression might cause a new local balance between vascular ET-1 and nitric oxide, resulting in no alterations of blood pressure.     

Acute hemodynamic and neurohumoral effects of selective ET(A) receptor blockade in patients with congestive heart failure. ET 003 Investigators

OBJECTIVES

To investigate the hemodynamic effects of the selective endothelin (ET)_A receptor antagonist LU135252 in patients with congestive heart failure (CHF).

BACKGROUND

Nonselective ET_A/B receptor antagonists improve hemodynamics in patients with CHF. Since ET_B receptors mediate the release of nitric oxide and the clearance of ET-1, selective ET_A antagonists are of special interest.

METHODS

The hemodynamic effects of a single oral dose of the selective ET_A receptor antagonist LU135252 (1, 10, 30, 100 or 300 mg) were investigated in a multicenter study involving 95 patients with CHF (New York Heart Association II–III) with an ejection fraction ≤35%.

RESULTS

Baseline ET-1 positively correlated with pulmonary vascular resistance, pulmonary capillary wedge pressure (PCWP), and mean pulmonary artery pressure (MPAP, r = 0.37–0.50, p < 0.0004) but were inversely related to cardiac index (CI; r = −0.36, p = 0.0004). LU135252 dose dependently increased CI and decreased mean arterial pressure and systemic vascular resistance (p < 0.03–0.0002), while heart rate remained constant or decreased slightly. Pulmonary capillary wedge pressure, MPAP, pulmonary vascular resistance and right atrial pressure also decreased significantly (p < 0.035–< 0.0001). Two hours after LU135252, plasma ET-1 did not significantly increase after 1 mg but did so by 23% (p = 0.003), 29% (p = 0.0018), 56% (p < 0.0001) and 101% (p < 0.0001) after 10, 30, 100 and 300 mg, respectively, while plasma catecholamines remained constant.

CONCLUSIONS

In patients with CHF, a single oral dose of the selective ET_A receptor antagonist LU135252 improves hemodynamics in a dose-dependent manner without activation of other neurohumoral systems and is well tolerated over a wide dose range.     

Acute hemodynamic and neurohumoral effects of selective ETA receptor blockade in patients with congestive heart failure

OBJECTIVESTo investigate the hemodynamic effects of the selective endothelin (ET)_A receptor antagonist LU135252 in patients with congestive heart failure (CHF).BACKGROUNDNonselective ET_A/B receptor antagonists improve hemodynamics in patients with CHF. Since ET_B receptors mediate the release of nitric oxide and the clearance of ET-1, selective ET_A antagonists are of special interest.METHODSThe hemodynamic effects of a single oral dose of the selective ET_A receptor antagonist LU135252 (1, 10, 30, 100 or 300 mg) were investigated in a multicenter study involving 95 patients with CHF (New York Heart Association II–III) with an ejection fraction ≤35%.RESULTSBaseline ET-1 positively correlated with pulmonary vascular resistance, pulmonary capillary wedge pressure (PCWP), and mean pulmonary artery pressure (MPAP, r = 0.37–0.50, p < 0.0004) but were inversely related to cardiac index (CI; r = −0.36, p = 0.0004). LU135252 dose dependently increased CI and decreased mean arterial pressure and systemic vascular resistance (p < 0.03–0.0002), while heart rate remained constant or decreased slightly. Pulmonary capillary wedge pressure, MPAP, pulmonary vascular resistance and right atrial pressure also decreased significantly (p < 0.035–< 0.0001). Two hours after LU135252, plasma ET-1 did not significantly increase after 1 mg but did so by 23% (p = 0.003), 29% (p = 0.0018), 56% (p < 0.0001) and 101% (p < 0.0001) after 10, 30, 100 and 300 mg, respectively, while plasma catecholamines remained constant.CONCLUSIONSIn patients with CHF, a single oral dose of the selective ET_A receptor antagonist LU135252 improves hemodynamics in a dose-dependent manner without activation of other neurohumoral systems and is well tolerated over a wide dose range.     

Increased pulmonary prostacyclin synthesis in rats with chronic hypoxic pulmonary hypertension

OBJECTIVE: The regulation of pulmonary prostacyclin synthesis is not completely understood. We tested the hypothesis that prostacyclin production is predominantly stimulated by hemodynamic factors, such as increased shear-stress, and is thus increased in rats with chronic hypoxic pulmonary hypertension. METHODS: To this end, we determined pulmonary prostacyclin synthase (PGIS) gene expression, circulating levels of the stable prostacyclin metabolite 6-keto prostaglandin F(1alpha) (6-keto-PGF(1alpha)), pulmonary endothelin (ET)-1 gene expression, and ET-1 plasma levels in rats exposed to 4 weeks of hypoxia (10% O(2)) in the presence or absence of either the nitric oxide (NO) donor molsidomine (MD, 15 mg/kg/day) or the ET-A receptor antagonist LU135252 (LU, 50 mg/kg/day). RESULTS: Right ventricular systolic pressure (RVSP), the cross-sectional medial vascular wall area of pulmonary arteries, and ET-1 production increased significantly during hypoxia. PGIS mRNA levels increased 1.7-fold, and 6-keto-PGF(1alpha) plasma levels rose from 8.2+/-0.8 to 12.2+/-2.2 ng/ml during hypoxia (each P<0.05 vs. normoxic controls). MD and LU reduced RVSP and pulmonary vascular remodeling similarly (each P<0.05 vs. hypoxia), but only MD inhibited pulmonary ET-1 formation (P<0.05 vs. hypoxia). Nevertheless, both drugs attenuated the increase in PGIS gene expression and plasma 6-keto-PGF(1alpha) levels (each P<0.05 vs. hypoxia). CONCLUSION: Our data suggest that prostacyclin production in hypertensive rat lungs is predominantly increased by hemodynamic factors while hypoxia, NO and ET-1 per are less important stimuli, and that this increase may serve as a compensatory mechanism to partially negate the hypoxia-induced elevation in pulmonary vascular tone.     

Angiotensin II and serotonin potentiate endothelin-1-induced vascular smooth muscle cell proliferation

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation induced by various growth factors has been implicated in a wide variety of pathological processes, including hypertension, atherosclerosis and restenosis after angioplasty. OBJECTIVES: To investigate the interactions among well-known potent vasoconstrictor substances, endothelin-1 (ET-1), angiotensin II (Ang II), and serotonin (5-HT), on VSMC proliferation. METHODS: Growth-arrested rabbit VSMCs were incubated with different concentrations of ET-1 in the absence or presence of Ang II, 5-HT, or both. VSMC proliferation was examined by increases in incorporation of [3H]thymidine into DNA and in cell number. RESULTS: ET-1, Ang II and 5-HT stimulated DNA synthesis in a dose-dependent manner. ET-1 had a maximal effect at a concentration of 0.5 micromol/l (259% of control), Ang II at 1 micromol/l (173%), and 5-HT at 50 micromol/l (205%). When added together, ET-1 (0.1 micromol/l) and Ang II (1 micromol/l) synergistically induced DNA synthesis (341%). When the vasoconstrictors were tested in combination, even non-mitogenic concentrations of ET-1 (0.01 nmol/l) potentiated 5-HT (5 micromol/l)-induced DNA synthesis (404%). Co-incubation of ET-1 (0.01 micromol/l) with Ang II (1 micromol/l) and 5-HT (5 micromol/l) synergistically induced DNA synthesis (566%). These effects on DNA synthesis were paralleled by an increase in cell number. The ETA/B non-selective receptor antagonist, TAK044 (1 micromol/l) and the ETA receptor antagonist, BQ123 (1 micromol/l), but not the ETB receptor antagonist, BQ788 (1 micromol/l), inhibited the mitogenic effect of ET-1 and its interaction with Ang II or 5-HT. In addition, TAK044 (1 micromol/l) or BQ123 (1 micromol/l) along with the angiotensin II type 1 (AT1) receptor antagonist, candesartan (1 micromol/l), the 5-HT2A receptor antagonist, sarpogrelate (10 micromol/l), or both, inhibited the interactions of ET-1 with Ang II or 5-HT. CONCLUSIONS: Our results suggest that Ang II and 5-HT could potentiate ET-1-induced VSMC proliferation. Inhibition of ETA, AT1, and 5-HT2A may be effective in the treatment of VSMC proliferative disorders associated with hypertension, atherosclerosis and restenosis after angioplasty.     

Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis

AIM： To determine the platelet-activating factor （PAF） synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis.
METHODS： Kupffer cells, isolated from the livers of control and CCl4-induced cirrhotic rats, were placed in serum-free medium overnight. PAF saturation binding, ET-1 saturation and competition binding were assayed. ET-1 induced PAF synthesis, mRNA expression of PAF, preproendothelin-1, endothelin A （ETA） and endothelin B （ETB） receptors were also determined.
RESULTS： A two-fold increase of PAF synthesis （1.42 ± 0.14 vs 0.66 ± 0.04 pg/μg DNA） and a 1.48-fold increase of membrane-bound PAF （1.02 ± 0.06 vs 0.69 ± 0.07 pg/μg DNA） were observed in activated Kupffer cells of cirrhotic rats. The application of ET-1 to Kupffer cells induced PAF synthesis in a concentration-dependent manner in both cirrhotic and normal rats via ETB receptor, but PAF synthesis in the activated Kupffer cells was more effective than that in the normal Kupffer cells. In activated Kupffer cells, PAF receptor expression and PAF binding capacity were markedly enhanced. Activated Kupffer cells raised the [^125I]-ET-1 binding capacity, but changed neither the affinity of the receptors, nor the expression of ETA receptor.
CONCLUSION： Kupffer cells in the course of CCl4- induced cirrhosis are the main source of increased PAF. ET-1 is involved endogenously in stimulating the PAF synthesis in activated Kupffer cells via ETB receptor by paracrine. ETA receptor did not appear in activated Kupffer cells, which may exacerbate the hepatic and extrahepatic complications of cirrhosis.     

Norepinephrine-induced aortic hyperplasia and extracellular matrix deposition are endothelin-dependent.

BACKGROUND: Sympathetic hyperactivity is observed in several disease states and may contribute to cardiovascular hypertrophic remodeling. Endothelin has been suggested to be a mediator of hypertrophy. OBJECTIVE: To examine the involvement of endothelin in maintaining the growth response induced by exogenous norepinephrine. DESIGN AND METHODS: Rats were treated with norepinephrine (2.5 microg/Kg per min subcutaneously) for 2 and 4 weeks, alone or in association with the selective endothelin-A (ETA) receptor antagonist, darusentan (LU135252, 30 mg/Kg per day orally) for weeks 3 and 4. RESULTS: Increases in medial cell number and accumulation of collagen and elastin characterized norepinephrine-induced aortic remodeling. These effects occurred without marked changes of mean arterial pressure, but may be related to enhanced pressure variability in addition to direct effects of norepinephrine. Inhibition of ETA receptors by darusentan reversed aortic alterations produced by infusion of norepinephrine. Evaluation of medial apoptosis did not reveal any significant change in any group at 4 weeks. CONCLUSIONS: Antagonism of ETA receptors effectively and rapidly reversed norepinephrine-induced aortic structural and compositional changes, suggesting a central role of endothelin in mediating this response. Thus, ETA receptor antagonists may help to regress large artery remodeling in conditions of increased circulating catecholamine concentrations.     

Identification of endothelin receptor subtypes in human renal cortex and medulla using subtype-selective ligands.

High affinity and high density endothelin (ET)-binding sites were identified in membranes prepared from human kidney cortex and medulla. Saturation binding experiments performed in membranes prepared from cortex and medulla using [125I]ET-1 and [125I]ET-3 revealed that the proportion of [125I]ET-3-binding sites was 30-35% less than that of [125I]ET-1-binding sites. The apparent dissociation constants and maximum binding for [125I]ET-1 and [125I]ET-3 to membranes from cortex were 91 +/- 5 pM and 165 +/- 10 fmol/mg protein, and 117 +/- 9 pM and 110 +/- 7 fmol/mg protein, respectively, whereas in medulla they were 139 +/- 10 pM and 360 +/- 11 fmol/mg protein, and 142 +/- 11 pM and 245 +/- 15 fmol/mg protein, respectively. In the presence of 10 nM sarafotoxin-6c, which is selective for ETB receptors, [125I]ET-1 binding was decreased by 65-70%, whereas [125I]ET-3 binding was totally abolished, suggesting that 65-70% of [125I]ET-1 binding and 100% of [125I]ET-3 binding was to ETB receptors. This was further confirmed by the use of a cyclic pentapeptide [cyclo(D-Trp,D-Asp,L-Pro, D-Val,L-Leu)] (BQ123), which is selective for ETA receptors. In the presence of 1 microM BQ123, [125I]ET-1 binding was decreased by 25-30%, whereas [125I]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ETA subtypes, and that [125I]ET-1 bound to both ETA and ETB receptors with the same high affinity, but [125I]ET-3 bound only to ETB receptors with high affinity. These results suggest that human kidney cortex and medulla contain ETA and ETB receptors in a ratio of 30:70, and that sarafotoxin-6c and BQ123 are valuable tools in identifying the subtype of ET receptors in various tissues.     

Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788.

We describe the characteristics of a potent and selective endothelin (ET) B-receptor antagonist, BQ-788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1- methoxycarbonyltryptophanyl-D-norleucine]. In vitro, this compound potently and competitively inhibits 125I-labeled endothelin 1 (ET-1) binding to ETB receptors on human Girardi heart cells (IC50, 1.2 nM) but only poorly inhibits the binding to ETA receptors on human neuroblastoma cell line SK-N-MC cells (IC50, 1300 nM). In isolated rabbit pulmonary arteries, BQ-788 shows no agonist activity up to 10 microM and competitively antagonizes the vasoconstriction induced by an ETB-selective agonist, BQ-3020 (pA2, 8.4). In rat, an ETA-selective antagonist, BQ-123 (1 mg/kg, i.v.), does not affect transient depressor response to ET-1 (0.3 nmol/kg, i.v.) but potently inhibits following sustained pressor response; vice versa, BQ-788 (1 mg/kg, i.v.) abolishes the depressor response, resulting in a rapid onset of apparently enhanced pressor response. Thus, being a potent and selective ETB receptor antagonist, BQ-788 may be considered as a powerful tool for investigating the role of ET in physiological and pathological processes.     

Role of endothelin-1 in the migration of human olfactory gonadotropin-releasing hormone-secreting neuroblasts

FNC-B4 neuroblasts that express both neuronal and olfactory markers have been established and cloned. These cells express GnRH and both the endothelin-1 (ET-1) gene and protein and respond in a migratory manner to GnRH in a dose-dependent manner. Previous research has shown that FNC-B4 cells produce and respond to ET-1 by regulating the secretion of GnRH through endothelin type A receptors and by stimulating their proliferation through endothelin type B (ETB) receptors. In this study, we found that FNC-B4 cells are able to migrate in response to ET-1 through the involvement of ETB receptors. Combined immunohistochemical and biochemical analyses showed that ET-1 triggered actin cytoskeletal remodeling and a dose-dependent increase in migration (up to 6-fold). Whereas the ETB receptor antagonist (B-BQ788) blunted the ET-1-induced effects, the ETA receptor antagonist (A-BQ123) did not. Moreover, we observed that FNC-B4 cells were independently and selectively stimulated by ET-1 and GnRH. We suggest that ET-1, through ETB receptor activation, may be required to maintain an adequate proliferative stem cell pool in the developing olfactory epithelium and the subsequent commitment to GnRH neuronal migratory pattern. The coordinate interaction between ET receptors and GnRH receptor participates in the fully expressed GnRH-secreting neuron phenotype.     

Role of endothelin in the hypertrophic remodeling of small arteries induced by exogenous norepinephrine]

In a subset of hypertensive patients, activity of the sympathetic nervous system (SNS) is enhanced. Hypertension is also associated with an adaptative process where small arteries (lumen < 300 microns) are subjected to structural changes (eutrophic or hypertrophic remodeling). Since, it has been shown that norepinephrine (NE) can induced proliferation of vascular smooth muscle cells, the purpose of the present study was to determine the effect of a chronic treatment with NE, mimicking hyperactivity of SNS, on small artery structure. The role of endothelin (ET) in the process was also evaluated. To achieve these goals, control rats were compared with rats receiving NE 2.5 micrograms/kg/min alone or in combination with LU135252 30 mg/kg/d (ET-receptor antagonist, affinity ETA/ETB approximately equal to 100) for 2 weeks. Blood pressure was measured intra-arterially in conscious rats prior to sacrifice. Geometric parameters of the basilar artery were determined in pressurized and perfused conditions with calcium free Krebs solution. Plasma NE and arterial mesenteric ET levels were determined by HPLC and RIA respectively. Blood pressured was not altered following exogenous administration of NE for 2 weeks. However, media thickness increased while the lumen diameter was reduced at the level of the basilar artery, leading to elevated media:lumen ratio (p < 0.05). This morphological alteration was associated with a significant augmentation of the basilar artery cross-sectional area (CSA). Co-administration of LU135252 with NE prevented partially the increase of M/L while the elevation of CSA was completely blunted. Plasma levels of NE were significantly and similarly elevated in groups receiving NE but, interestingly, mesenteric ET levels were not modified by any treatment. These results suggest that chronic NE administration induced an hypertrophic inward remodeling of small arteries independently from blood pressure, which required the participation of ET as an obligatory intermediate. Furthermore, the local production of ET is probably enhanced transiently in the first days of NE administration and come back to control level at 2 weeks. Thus, early therapy initiation with an ET-receptor antagonist prevents vascular remodeling in conditions of SNS hyperactivity, which may contribute to lower risks of end-organ damage.     

Collagen accumulation after myocardial infarction: effects of ETA receptor blockade and implications for early remodeling

OBJECTIVES: Endothelin A (ETA) receptor blockade started early after myocardial infarction (MI) promotes adverse left ventricular (LV) dilatation. We tested the hypothesis that inhibition of ETA receptors during the early phase of healing affects collagen synthesis and accumulation, and induces expansion of infarcted myocardium. METHODS: Starting 3 h after coronary ligation, female Wistar rats were treated with the selective ETA receptor antagonist LU 135252 (30 mg/kg body wt/day) or placebo. A period of 7 days after MI, hemodynamic, morphometric and biochemical studies were performed. RESULTS: ET(A) receptor blockade enhanced infarct expansion index and decreased LV systolic function. Infarct scar of LU 135252-treated rats displayed decreased gene expression of fibrillar type I/III collagens and of transforming growth factor-beta(1) (TGF-beta(1)). Collagen content in the infarct scar and border regions was lower after ETA inhibition. In addition, Western blot analysis revealed, after ETA receptor blockade, enhanced matrix metalloproteinases MMP-13, and MMP-2 expression in the infarcted LV myocardium. CONCLUSIONS: These data demonstrate that endothelin stimulates collagen accumulation at the site of infarction. Decreased collagen and TGF-beta(1) gene expression, associated with enhanced infarct expansion and MMP up-regulation likely contributes to ETA receptor blockade-mediated deleterious effects on ventricular remodeling after infarction.     

Endothelin-1[1-31]: a novel autocrine-paracrine regulator of human adrenal cortex secretion and growth.

Endothelin (ET)-1[1-21] stimulates steroid secretion and zona glomerulosa growth and is expressed in the human and rat adrenal cortex together with its receptor subtypes A and B (ETA and ETB). Although ET-1[1-21] is generated from bigET-1 by an ET-converting enzyme (ECE-1), there is evidence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, the role of which in adrenal pathophysiology is largely unknown. Gene expression and immunohistochemical studies allowed localization of chymase in the normal human adrenal cortex. Sizable amounts, not only of ET-1[1-21] but also of ET-1[1-31], were found in the adrenal vein plasma of three patients. ET-1[1-21] and ET-1[1-31] elicited a clear-cut secretory response by dispersed human adrenocortical cells, ET-1[1-31] being significantly less potent than ET-1[1-21]. The secretagogue effect of ET-1[1-31] was abolished by the ETA receptor antagonist BQ-123 and was unaffected by the ETB receptor antagonist BQ-788. Because, in humans, the secretagogue effect of ET-1[1-21] involves both ETA and ETB receptors, the weaker action of ET-1[1-31] could be attributable to a selective ETA receptor activation. Two lines of evidence support this contention: 1) ET-1[1-31] was more effective than ET-1[1-21] in stimulating ETA-mediated cell proliferation of human adrenocortical cells cultured in vitro; and 2) autoradiography showed that a) ET-1[1-31] displaced in vitro [(125)I]ET-1[1-21] binding to the ETA, but not ETB receptors, in human internal thoracic artery rings; and b) BQ-123, but not BQ-788, eliminated [(125)I]ET-1[1-31] binding in the rat adrenal cortex.     

Stimulation of the renin-angiotensin system by endothelin subtype A receptor blockade in conscious dogs.

Previous studies in dogs have shown additive or even synergistic effects of combined angiotensin-converting enzyme inhibition and either nonselective endothelin subtype A/B (ETA/B) or selective endothelin subtype A (ETA) receptor blockade on renal vascular resistance and mean arterial blood pressure. A possible mechanism underlying this interaction may be a stimulation of the renin-angiotensin system during endothelin (ET) receptor blockade. We therefore investigated whether plasma renin activity and renin release are regulated by the ETA receptor. Experiments were made in conscious, chronically instrumented dogs receiving either saline or the selective ETA receptor antagonist LU 135252 (10 mg/kg IV). Eighty to 100 minutes after the administration of LU 135252 (n=5), heart rate (99+/-7 versus 81+/-6 bpm; P<0.05) and renal blood flow (327+/-40 versus 278+/-36 mL/min; P<0.05) were increased significantly, whereas mean arterial blood pressure tended to be lower (93+/-5 versus 105+/-7 mm Hg). These changes were associated with a 2-fold increase in plasma renin activity (0.74+/-0.12 versus 0.37+/-0.10 ng angiotensin I per milliliter per hour; P<0.05). Measurements of renin release at various renal perfusion pressures (n=5) with the use of a vascular occluder implanted around the left renal artery revealed that ETA receptor blockade did not alter renin release at resting renal perfusion pressure (78+/-25 versus 71+/-39 U/min) but strongly enhanced the sensitivity of pressure-dependent renin release <80 mm Hg approximately 2.2-fold. In conclusion, selective ETA receptor blockade is associated with a stimulation of the circulating renin-angiotensin system, which results from both a sensitization of pressure-dependent renin release and a larger proportion of blood pressure values falling into the low pressure range, where renin release is stimulated. These find-ings strengthen the view that ET and the renin-angiotensin system closely interact to regulate vascular resistance and provide a physiological basis for synergistic hypotensive effects of a combined blockade of both pressor systems.