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Although activation of the endothelin (ET) system contributes to pulmonary hypertension, modifications of the cardiopulmonary ET system and its responses to chronic ET receptor blockade are not well known. To investigate this, rats were injected with monocrotaline (60 mg/kg intraperitoneal) or saline, followed with treatment with the selective ETA receptor antagonist LU135252 (LU; 50 mg·kg-1·day-1) or with saline. After 3 weeks, haemodynamics, cardiac hypertrophy, ET-1 levels and cardiopulmonary ET-receptor-binding profile were evaluated. Monocrotaline (n=7) elicited marked pulmonary hypertension and right ventricular hypertrophy compared with controls (n=8). Both variables were substantially attenuated by LU therapy (n=8; P<0.05 for both). After monocrotaline, right ventricular ET-1 levels were more significantly increased than in the left ventricle (+198% compared with +127%; P<0.05). ETB receptor density was augmented (3-fold) in the right ventricle, whereas that of ETA receptors was not affected. LU treatment also significantly attenuated these alterations (P<0.05). In the lungs, ET-1 levels were not increased after monocrotaline, whereas the balance of ETB to ETA receptors was altered, with a trend toward a lower percentage of ETB than in the control rats. LU treatment did not affect these variables in the lungs. Therefore monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy are associated with the up-regulation of ET-1 and ETB receptors in the right ventricle. These alterations are attenuated with the reduction of pulmonary hypertension and right ventricular hypertrophy after chronic blockade of the ETA receptors, supporting the role of the ET system in right ventricular hypertrophy.

Cardiac endothelin-1 (ET-1) levels and ET receptor expression are increased in congestive heart failure (CHF). In order to determine whether this results in increased responsiveness of ET-A or ET-B receptors to ET-1, we evaluated the contractile effects of ET-1 in isolated papillary muscles isolated from hearts of control rats and from rats 4 weeks post myocardial infarction (MI) having received no therapy or chronic quinapril therapy. The ET-1 dose-response was biphasic in normal muscles. The use of the selective ET-A receptor antagonist BQ123 and the selective ET-B receptor antagonist BQ788 revealed that the initial decrease in tension was the result of ET-B receptor stimulation. Blockade of nitric oxide (NO) production with L-NAME abolished the initial decrease in tension. MI resulted in CHF that was partially reversed by quinapril. In MI, the positive inotropic effects of ET-1 were enhanced due to the loss of the initial ET-B receptor mediated decrease in tension, as well as an increase in the positive inotropic effects of ET-A receptors. This was associated with an increase in ET-A and ET-B receptor mRNA and a decrease in cardiac ecNOS protein. Four weeks of therapy with quinapril attenuated the positive inotropic effects of ET-1 and prevented the increase in ET-A receptor mRNA. Although quinapril did not restore the effects of ET-B receptor stimulation or prevent the increase in ET-B mRNA, it did restore cardiac ecNOS protein expression. Thus, the inotropic response to ET-1 is biphasic due to an overall positive inotropic effect of ET-A receptor stimulation and an ET-B receptor mediated decrease in contractility at low ET-1 concentrations which appears to be mediated by cardiac ecNOS (NO). In post-MI CHF, responsiveness to ET-A receptors increases and the ET-B mediated negative inotropic response is lost despite an increase in both receptor subtypes. Quinapril therapy attenuates these effects and normalises cardiac ecNOS protein.

Endothelin1 ET1 levels are markedly increased in sepsis. Since ET1 is primarily transcriptionally regulated, there should be a corresponding increase in preproendothelin1 ppET1. Our objective was to determine whether ppET1 is increased in pigs with a low systemic vascular resistance. We also examined the distribution of ET1 and the regulation of endothelinconverting enzyme 1 ECE1, the rate limiting enzyme in ET1 production. We anesthetized and ventilated 16 pigs. We measured arterial, pulmonary, and central venous pressures, as well as cardiac output. ET1 was measured by radioimmunoassay in plasma and in multiple tissues. We infused 20 gkg of endotoxin over 2 h and then sacrificed the animals. ppET1 and ECE1 mRNA were assessed by Northern analysis. We performed immunohistochemistry for the assessment of tissue ET1 and ECE1. The systemic vascular resistance rose at 30 min, but fell by 120 min. Plasma ET1 more than doubled by 2 h. However, there was no change in the concentration of ET1 in any tissue except in the pulmonary artery. By immunohistochemistry, there was also no change in ET1 in aorta, vena cava, heart, lung, liver, and kidney. Distribution of ECE1 followed that of ET1 on immunohistochemistry. There was a significant increase in ppET1 mRNA in liver, kidney papillae, and vena cava, and a tendency for an increase in other tissues. This was paralleled by an increase in ECE1 mRNA. In conclusion, the amount of ECE1 mRNA and protein parallel those of ET1. Endotoxemia is associated with a marked increase in plasma ET1 and an increase in ppET1 and ECE1 mRNA in multiple tissues however, there was no significant change in tissue ET1 except in the pulmonary artery. The rise in plasma levels without a change in tissue levels suggests a greater release into the vasculature in sepsis than under normal conditions.

Measurements of pulmonary pressure and resistance are still considered to be the “gold standard” in the evaluation of pulmonary hypertension (PH), despite their limitations in predicting irreversible disease. Hemodynamic assessment also only provides a global evaluation of the pulmonary vascular bed, whereas PH is an inhomogeneous disease of the vessel wall.

We hypothesized that endothelin (ET) release during exercise may be triggered by α-adrenergic-receptor activation and thereby influence coronary hemodynamics and O2metabolism in dogs. Exercise resulted in coronary blood flow increases (to 1.88 ± 0.26 from 1.10 ± 0.12 ml · min−1· g−1) and in a fall (P< 0.01) in coronary sinus O2saturation (17.4 ± 1.5 to 9.6 ± 0.7 vol%), whereas myocardial O2consumption (MV˙o2) increased (109 ± 13% from 145 ± 16 μl O2· min−1· g−1). Tezosentan, a dual ETA/ETB-receptor blocker, slightly reduced mean arterial pressure (MAP) and increased heart rate throughout exercise. The relationship between coronary sinus O2saturation and MV˙o2was shifted upward (P< 0.05) after tezosentan administration; i.e., as MV˙o2increased during exercise, coronary sinus O2saturation was disproportionately higher after ET-receptor blockade. After propranolol, tezosentan resulted in significant decreases (P< 0.05) in left ventricular pressure, the first derivative of left ventricular pressure over time, and MAP during exercise. As MV˙o2increased during exercise, coronary sinus O2saturation levels after tezosentan became superimposable over those observed before ET-receptor blockade. Thus dual blockade of ETA/ETBreceptors alters coronary hemodynamics and O2metabolism during exercise, but ET activity failed to increase beyond baseline levels.

Pulmonary hypertension is associated with endothelial dysfunction that may mediate or contribute to the disease process; among those abnormalities is an increase in circulating endothelin-1 levels. We investigated the effect of the orally active endothelin A receptor antagonist LU 135252 (LU) on the development of monocrotaline (MCT)-induced pulmonary hypertension and endothelial metabolic dysfunction. Rats were assigned to four groups by receiving a single dose of MCT or saline, followed by once-daily gavage with LU (50 mg/kg) or saline for 3 weeks. Plasma immunoreactive endothelin-1 levels doubled after MCT and were unaffected by LU therapy. The MCT-induced increase in right ventricular systolic pressure (72.5 ± 15.9 mmHg) and hypertrophy (right ventricle/[left ventricle plus septum weight]; 0.58 ± 0.08) were reduced by LU to 42.7 ± 8.5 mmHg (P < .01) and 0.42 ± 0.05 (P < .01), respectively. LU, however, did not modify MCT-induced pulmonary artery medial hypertrophy. Pulmonary vascular endothelial metabolic activity was evaluated in isolated lungs by measuring endothelium-bound angiotensin-converting enzyme activity using a synthetic angiotensin-converting enzyme substrate,3H-benzoyl-phenylalanly-glycyl-proline. MCT reduced fractional 3H-benzoyl-phenylalanly-glycyl-proline hydrolysis (0.488 ± 0.051, P < .01) which was normalized by LU therapy (0.563 ± 0.050). LU treatment alone had no significant effect on any of these parameters. We conclude that the endothelin A antagonist LU reduces MCT-induced pulmonary hypertension and right ventricular hypertrophy and restores endothelial metabolic function. These results support the development of endothelin antagonists for the treatment of pulmonary hypertension and associated endothelial metabolic abnormalities.

Pulmonary hypertension is associated with endothelial dysfunction that may mediate or contribute to the disease process; among those abnormalities is an increase in circulating endothelin-1 levels. We investigated the effect of the orally active endothelin A receptor antagonist LU 135252 (LU) on the development of monocrotaline (MCT)-induced pulmonary hypertension and endothelial metabolic dysfunction. Rats were assigned to four groups by receiving a single dose of MCT or saline, followed by once-daily gavage with LU (50 mg/kg) or saline for 3 weeks. Plasma immunoreactive endothelin-1 levels doubled after MCT and were unaffected by LU therapy. The MCT-induced increase in right ventricular systolic pressure (72.5 +/- 15.9 mmHg) and hypertrophy (right ventricle/[left ventricle plus septum weight]; 0.58 +/- 0.08) were reduced by LU to 42.7 +/- 8.5 mmHg (P < .01) and 0.42 +/- 0.05 (P < .01), respectively. LU, however, did not modify MCT-induced pulmonary artery medial hypertrophy. Pulmonary vascular endothelial metabolic activity was evaluated in isolated lungs by measuring endothelium-bound angiotensin-converting enzyme activity using a synthetic angiotensin-converting enzyme substrate, 3H-benzoyl-phenylalanly-glycyl-proline. MCT reduced fractional 3H-benzoyl-phenylalanly-glycyl-proline hydrolysis (0.488 +/- 0.051, P < .01) which was normalized by LU therapy (0.563 +/- 0.050). LU treatment alone had no significant effect on any of these parameters. We conclude that the endothelin A antagonist LU reduces MCT-induced pulmonary hypertension and right ventricular hypertrophy and restores endothelial metabolic function. These results support the development of endothelin antagonists for the treatment of pulmonary hypertension and associated endothelial metabolic abnormalities.

1. There is an increase in circulating levels of vasoconstrictive hormones and an alteration in baroreceptor responsiveness with aging. The role of changes in endothelium-dependent and -independent vasoconstrictive hormones in relation to age and gender, with simultaneous assessment of autonomic balance in response to head-up tilt, has been incompletely studied. 2. Sixteen young [25 ± 3 years (mean ± SEM)] and 16 older normal volunteers (68 ± 7 years) underwent a 30 min head-up tilt test at 60°. Haemodynamics were measured every 5 min and blood samples for neurohormone measurement were drawn at baseline, 5, 10, 15 and 30 min into the test. Heart rate variability was analysed in 5 min segments at the baseline, and during the test. The younger subjects exhibited a greater increase in heart rate and diastolic blood pressure, despite lower absolute levels of noradrenaline (norepinephrine) and endothelin-1. Analysis of heart rate variability yielded a decrease in both high- and low-frequency bands in the aged; power at low-frequency decreased only in the young subjects. The age-related differences in blood pressure and noradrenaline levels were markedly attenuated in the female subjects. In addition, endothelin-1 levels and power spectral measurements at low frequency were the lowest in younger females throughout the tilt. 3. Despite attenuated cardiovascular response to tilt, both systemic adrenergic ‘drive’ and endothelin-1 levels increase in parallel with aging. Thus, endothelium-dependent and -independent vasoconstrictive hormone levels increase with age in the resting state and in response to neurohumoral stimulation in humans.

Objective Pulmonary hypertension (PHT) is associated with increased endothelin-1 (ET-1) levels that correlate with the severity of the disease. The pulmonary circulation is an important site for ET-1 metabolism and may modulate plasma ET-1 through an increase in production, a reduction in removal, or a combination of both. We measured and compared pulmonary metabolism of circulating ET-1 in controls and in patients with PHT. Methods and Results The indicator-dilution technique was combined with measurements of ET-1 levels to quantify pulmonary metabolism of ET-1 in controls (n = 13) and in patients with PHT (n=17). ET-1 levels doubled in PHT (p < 0.05) and, although there was no difference between aortic and pulmonary artery levels in controls (0.68 +/- 0.09 and 0.61 +/- 0.08 pg/ml, respectively, p=0.22), they tended to be higher in PHT (1.23 +/- 0.26 vs 1.07 +/- 0.19 pg/ml, p=0.08). Pulmonary extraction of tracer iodine-125-ET-1 was reduced from 47% +/- 2.0% in the controls to 34% +/-3.6% in PHT (p = 0.005) and inversely correlated with the severity of pulmonary hypertension (r = -0.524, p = 0.03). Consequently, circulating ET-1 clearance was reduced by PHT from 1424+/- 77 ml/min to 892 +/- 119 ml/min (p < 0.001). Pulmonary production of circulating ET-1 (in picograms per minute) was not different but the quantity of ET-1 that survives passage through the lungs was increased by PHT (1860 +/- 359 pg/min vs 992+/-152pg/min, p = 0.037). Conclusion PHT is associated with a reduced pulmonary clearance of ET-1 that contributes to the increase in circulating levels. (Am Heart J 1998;135:614-20.)

Endothelin is a novel endothelium-derived vasoactive peptide with potent vasoconstrictor action in the coronary bed; however, its possible contribution to myocardial ischemia and infarction is not known. Plasma endothelin-1 concentration was measured with use of a radioimmunoassay in serial venous samples from 22 patients over a 72 h period after acute myocardial infarction (14 patients with uncomplicated infarction [group I] and 8 patients with hemodynamic or ischemic sequelae [group II]). Twenty-two normal subjects and seven patients with stable angina served as the control subjects.

Endothelins (ETs) were initially characterized as potent vasoactive peptides acting through at least two distinct receptors, ETA and ETB. Subsequently, their significant growth- and hypertrophy-promoting properties in cardiac and other cells were recognized. We investigated the expression of endothelin receptors during differentiation of a pluripotential embryonal carcinoma cell line (P19) to a cardiomyocyte or a neural lineage. These cells resemble those of the inner cell mass of the blastocyst, and their differentiation is believed to closely mimic critical events in early embryogenesis. Differentiation of P19 to a cardiomyocyte lineage, by aggregation and exposure to dimethyl sulfoxide resulted in induction of ETA receptors as demonstrated by radioligand binding studies, Northern blotting, and reporter gene analysis. Moreover, the P19 differentiated to a cardiac lineage responded to ET-1 with a 3-fold increase in the secretion of atrial natriuretic peptide. In contrast, differentiation to a neural lineage, by aggregation and exposure to retinoic acid, was associated with the induction of predominantly ETB. Therefore, selective differentiation of the P19 led to the differential expression of endothelin receptors in a pattern consistent with that observed in normal myocardial and neural tissue. The induction of endothelin receptors in a model system of early embryogenesis provides strong support for the critical role of this peptide/receptor family in differentiation and development. As well, this model system is well suited for the study of the mechanisms controlling endothelin receptor expression during differentiation.

Lt has been proved repeatedly that the activity of a part of the cerebral cortex influences the homologous part of the cortex on the opposite side. Bykov1 in 1925 reported that the conditioned salivary reflex to tactile stimuli of a certain point of the skin in the dog may be obtained also from the symmetrical point of the opposite side of the body. In the visual sphere Myers and Sperry,2-5 by ingenious experiments in the cat, recently demonstrated that the ability for pattern discrimination acquired by the visual cortex of one hemisphere is also present in the visual cortex of the opposite hemisphere. The experiments of Burešová and Bureš6 gave similar results with other methods. Stamm and Sperry7 report analogous observations in the somesthetic sphere. Dawson,8,9 stimulating sensory nerves, registered cortical evoked potentials both in the homologous and in the opposite senory region of the

Objective: We evaluated the direct effects of long-term blockade of ET_A and ET_B receptors using a mixed endothelin (ET) receptor antagonist, LU224332, in the low density lipoprotein receptor (LDL-R) knockout mouse model of atherosclerosis. Methods: Four groups of LDL-R deficient mice were studied: control mice fed normal chow (group I); mice fed a high cholesterol (HC, 1.25%) diet alone (group II), HC fed animals treated with LU224332 (group III); and mice fed normal chow treated with the LU compound (group IV). All treatments were continued for 8 weeks at which time the animals were sacrificed and the aortae were removed and stained with oil red O. Atherosclerotic area (AA) was determined by quantitative morphometry and normalized relative to total aortic area (TA). Results: Cholesterol feeding resulted in a marked increased in total plasma cholesterol (15 fold) and widespread aortic atherosclerosis (AA/TA: group I: 0.013±0.007; group II: 0.33±0.11; P<0.001). Atherosclerotic lesions were characterized by immunohistochemistry as consisting mainly of macrophages which also showed high levels of ET-1 expression. Treatment with ET antagonist significantly reduced the development of atherosclerosis (AA/TA: group III: 0.19±0.07, P<0.01 vs. group II), without altering plasma cholesterol levels and blood pressure. The direct effect of LU224332 on macrophage activation and foam-cell formation was determined in vitro using a human macrophage cell line, THP-1. Treatment of the THP-1 cells with LU224332 significantly reduced cholesterol ester and triacylglycerol accumulation and foam-cell formation on exposure to oxidized LDL (P<0.01 and P<0.05, respectively). Conclusion: We conclude that a nonselective ET receptor antagonist substantially inhibited the development of atherosclerosis in a genetic model of hyperlipidemia, possibly by inhibiting macrophage foam-cell formation, suggesting a role for these agents in the treatment and prevention of atherosclerotic vascular disease.

Objectives: Plasma endothelin-1 (ET-1) increases in congestive heart failure (CHF). The pulmonary vascular bed could contribute to this increase through a reduced clearance. We evaluated the effect of tachycardia-induced CHF on pulmonary ET-1 kinetics. To discern between changes due to variations in pulmonary hemodynamics from true alterations of endothelial cell functions, we quantified ET-1 kinetics in isolated rat lungs under variable pressure and flow-rate conditions. Methods and Results: Indicator-dilution studies were performed in anesthetized dogs (n=14) before and 3 weeks after rapid ventricular pacing and in isolated lungs from healthy rats (n=4). In isolated lungs, graded increases in perfusion rate from 5–25 ml/min caused gradual reductions in ET-1 extraction from 60±1.5% to 17±4.9% (mean±S.D.). The capacity to clear ET-1 from the circulation, as computed from the permeability–surface area product (PS), however did not vary over this range of flows. CHF increased plasma ET-1 (11.2±11.4 vs. 5.2±1.6 fmol/ml, p<0.01), did not affect pulmonary ET-1 extraction (29.4±12.5% vs. 29.9±12.9%), but decreased the PS (8.3±5.4 cm3/s vs. 14.4±9.9 cm3/s, p=0.038). Contrary to the invariability of the PS in normal isolated rat lungs, CHF was associated with a positive relationship between the PS and pulmonary plasma flow (r=0.65, p<0.01). ET-1 binding studies in lung tissues showed no significant variations in ETA and ETB receptors densities but revealed a threefold decrease in binding affinity (p<0.01) that may explain the reduced clearance. Conclusion: CHF causes a reduction of pulmonary ET-1 clearance that likely contributes to the increased circulating ET-1 levels and reflects pulmonary metabolic dysfunction associated with this condition.