Author: gerald kurtulaj
Date: 28/03/2012




A turning point in the quest to identify endothelium-derived contracting factors was the discovery that, besides superoxide anions, endoperoxides and thromboxane A2, endothelial cells in culture synthesize and secrete a vasoconstrictor peptide .A gene coding for this peptide was then cloned and the product of this gene, endothelin-1 (ET-1), a peptide of 21-amino acid residues, was identified as one of the most potent known vasoconstrictors. Soon after, two other closely related peptides, ET-2 and ET-3, coded by two different genes, were characterized.Nevertheless, ET-1, an autocrine, paracrine and endocrine factor, is the most abundant and important endothelin isoform produced by endothelial cells. However, endothelins are also produced by a wide range of tissues, and indeed cells other than the endothelial cells are likely to be involved in the major role that these peptides play in development and/or physiology.


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ET-1 is synthesized as an inactive pre-proET-1 that undergoes removal of a short signal sequence by a signal peptidase to yield proET-1. In endothelial cells, proET-1 is cleaved by furin or PC7 convertases to form Big-ET-1. The basically inactive Big-ET-1 is then converted to the 21-amino acid ET-1, predominantly by the action of endothelin-converting enzymes (ECEs), integral membrane zinc peptidases belonging to the neprylisin family, which also includes the neutral endopeptidase 24-11 (NEP) and the Kell blood group family of proteins .Two different ECEs encoded by two different genes, ECE-1 and ECE-2 can generate ET-1, ET-2 and ET-3.
ECE-1 is constituted by 4 isoforms due to alternative splicing, which confers a different cellular localization for each of these different isoenzymes .In various endothelial cells, ECE-1d is the isoform predominantly expressed .In endothelial cells, ECE-1a and ECE-1c are preferentially addressed to the cell membrane, while ECE-1b and ECE-1d are preferentially located in endosomal compartments near the trans-Golgi network. Since Big-ET-1 can be present in the plasma, its conversion can occur both extracellularly and intracellularly. However, the ET-1 production by endothelial cells appears to be predominantly an intracellular process. In order to be active, ECE forms homodimers and heterodimers, which regulate the localization of the enzyme.This sub-cellular ECE-1 distribution is a dynamic process and ECE-1 is permanently recycled between the plasma membrane and the endosomal vesicles. In this latter compartment, ECE degrades various peptides which have been internalized with their receptors, for instance, tachykinins, somatostatin-14 and CGRP. ECE-1 disrupts the complex receptor–β-arrestins and regulates receptor recycling to the membrane.
ECE-2 is a membrane-bound, phosphoramidon sensitive zinc-metalloproteinase with an acidic pH optimum with no activity at pH 7.0, indicating that its activity is intracellular and intravesicular. It shares 60% homology with ECE-1 and also consists of four isomers.Besides the conversion of endothelins, ECE-2 and, to a lesser extent, ECE-1 are involved in the degradation of amyloid peptides.
Additionally, some Kell blood group proteins can generate ET-3. However, unlike ECE-1 and ECE-2 that preferentially generate ET-1, Kell proteins have a strong preference for big ET-3 and have much less activity versus big ET-1 and big ET-2.
The disruption of ECE-1 is lethal and is associated with craniofacial and gut defects (a phenotype similar to both ET-1/ETA- and ET-3/ETB-deficient mice. However, both ET-1 and ET-2 are still detected in the embryos (50% of the wild type).In contrast, the ECE-2 KO mice are healthy and fertile, but the double ECE-2/ECE-1 knockout mice show a more severe phenotype than the ECE-1 mice. Again, in these double knockout mice, the ET-1 production is not abolished and still represents 50% of that observed in wild-type animals.These data indicate that ECE-1 is responsible for the generation of the three peptides and is the predominant, but not the exclusive, enzyme involved in the generation of endothelins. Indeed, chymase and matrix metalloproteinases are also involved in the production of ET intermediates, such as ET-1-(1–31) and ET-1-(1–32), respectively.
Endothelins are degraded at least in part by NEP and deamidase and are rapidly eliminated from the circulation in the lungs, by binding to the endothelin ETB receptor subtype, here acting as a clearance receptor.

mRNA synthesis
protein synthesis

Endothelin synthesis is regulated by physicochemical factors such as pulsatile stretch, shear stress, and pH. Exercise upregulates myocardial ET-1 expression, which suggests ET-1 may play a role in maintaining cardiac function. Hypoxia is a strong stimulus for ET-1 synthesis that may be important in ischemia. ET-1 biosynthesis is stimulated by cardiovascular risk factors such as elevated levels of oxidized LDL cholesterol,and glucose, estrogen deficiency, obesity, cocaine use, aging, and procoagulant mediators such as thrombin. Furthermore, vasoconstrictors, growth factors, cytokines, and adhesion molecules also stimulate ET production . Inhibitors of ET-1 synthesis include nitric oxide (NO), prostacyclin, atrial natriuretic peptides, and estrogens.

The endothelins. ET: endothelin; ECE: endothelin-converting enzyme..

post-translational modifications


cellular localization,
biological function

Endothelin Receptors.

Endothelins interact with two G-protein-coupled receptors termed ETA and ETB .ET-1 is the preferential ligand for the ETAreceptor subtype, while the three peptides show a similar affinity toward the ETB receptor subtype .The ETA receptor is widely distributed and can be expressed in virtually every cell type of the body, but is especially predominant in vascular smooth muscle cells. The distribution of the ETB receptor is restricted to the cardiovascular and pulmonary systems, neurons, bone, pancreas and kidneys, but its expression is elevated in endothelial cells and renal tubules.
ET-1, beyond its function as a vasoactive peptide, also plays a crucial role in the atherogenic process by enhancing mitogenesis, inducing extracellular matrix formation and contributing to the development of inflammation within the vessel wall.Both ETAand ETB receptors are localized on vascular smooth muscle cells where they induce their vasoconstrictor, proliferative and hypertrophic action, but in arteries, the ETA receptor is the predominant vasoconstrictor receptor.
Endothelin-1 and regulation of vascular tone. Endothelin-1 (ET-1) is a vasoconstrictor predominantly by activating the endothelin A receptor subtype (ETA). It can also produce vasodilatation by stimulating the endothelin ETB receptor.

Endothelin-1 and regulation of vascular tone.

The contractions elicited by ETA receptor activation are unusual when compared to those produced by most other agonists as they are slowly developing and long lasting even after washing out the peptide. The almost irreversible binding of the peptide to this receptor and its persistent association even after internalization are likely to explain, at least in part, this prolonged signalling.The signal transduction following ETA receptor stimulation involves several G-protein-dependent and -independent pathways, including phospholipase C, phospholipase A2, adenylate cyclase, Rho kinase, transactivation of receptor tyrosine kinases, beta-arrestin and mitogen activated protein kinase cascades. The G-protein-dependent activation of phospholipase C and phospholipase A (predominantly Gq/11 and G12/13) leads to the usual cascade of events, i.e., on the one hand, the formation of IP3 and diacylglycerol and the resulting activation of protein kinase C, and on the other hand, the metabolism of arachidonic acid. The G-protein-independent pathway involves a direct interaction with β-arrestin, leading to the src/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway (src/ERK/MAPK). These pathways contribute to the increase in [Ca2+]i, linked to the facilitation of calcium influx and calcium mobilization, and the changes in calcium sensitivity, both being essential for ET-1-induced contraction (and proliferation) of vascular smooth muscle cells.

Endothelin-induced signal-transduction pathways.

Binding of ET-1 (or ET-2) to the ETA receptor in the plasma membrane triggers signal-transduction pathways through Gq, a pertussis toxin-insensitive G protein that is coupled to the ETA intracellular domain. Activation of phospholipase C (PLC), protein tyrosine kinases (PTKs; such as FAK), and RAS ultimately results in activation of the RAF/MEK/MAPK pathway. Mobilization of intracellular calcium (Ca2+), activation of protein kinase C (PKC) and activated MAPK induce nuclear transcription of protooncogenes (such as c-FOS, c-MYC and c-JUN), leading to cell growth and mitogenesis. Further analysis of the signalling pathway showed that ET-1 stimulated phosphatidylinositol 3-kinase (PI3K)-mediated AKT activation (not shown). DG, diacylglycerol; InsP3, inositol 1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; p125FAK, focal adhesion kinase. Modified from Ref. 6.
In the vascular wall, the ETB receptor is predominantly expressed in the endothelial cells and, beyond its role as a clearance receptor, its activation is associated with an increase in [Ca2+]i and leads to the release of the potent vasodilators and anti-proliferative factors, NO and prostacyclin .Both in vivo and in vitro, these endothelial effects of ET-1 counterbalance the effects of ETA stimulation on the vascular smooth muscle cells. However, in numerous cardiovascular diseases, this response is impaired and is associated with endothelial dysfunction, leaving the ETA-dependent responses unimpeded.In vascular smooth muscle cells, the activation of ETB receptors produces vasoconstriction, the signaling pathway being similar to that described for the ETA receptor.
However, besides the endothelial cells, in the renal collecting duct, the ETB receptor is highly expressed and plays a major role in regulating sodium excretion and therefore arterial blood pressure.


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Pathophysiological Action of Endothelins

Occlusive Vascular Disease


Hypercholesterolemia leads to endothelial dysfunction and is associated with increased ET levels in plasma and tissue.Oxidized LDL induces ET-1 gene expression in endothelial cells and the proliferation of vascular smooth muscle cells via ETA receptors. In addition, the increased release of ET-1 stimulates the synthesis of transforming growth factor-ß1, basic fibroblast growth factor, epiregulin, platelet-derived growth factor, and various adhesion molecules implicated in atherogenesis. ET-1 also increases neutrophil and platelet adhesion, thereby promoting lesion growth and coronary thrombosis. In experimental hypercholesterolemia, ETA receptor blockade reduced macrophage infiltration in fatty streaks. In hypercholesterolemic pigs, impaired endothelium-dependent vasodilatation is improved after ET receptor blockade.
In apolipoprotein E–deficient mice, ET-1 is involved in atherogenesis.Long-term ETA blockade reduces the extent of atherosclerosis, without affecting blood pressure or plasma cholesterol; it also restores NO-mediated endothelium-dependent relaxation and prevents increased vascular ET-1. ET-1 also contributes to myocardial infarction in mice with atherosclerosis.ET receptor blockade is also effective in reducing ischemic brain injury and vasospasm, 2 major factors determining the severity of stroke and its sequelae.

Effects of chronic ETA receptor blockade in experimental atherosclerosis.

"2.Coronary Artery Disease"

In atherosclerotic human arteries, ETA receptor mRNA is downregulated,while the binding capacity of ETA receptors is increased in atherosclerotic mice.In patients with angina pectoris but normal angiograms and in those with coronary artery disease and acute myocardial infarction,ET-1 plasma levels are increased. In human atherosclerotic lesions, the expression of ET-1 and ECE is enhanced. A functional role for tissue ET-1 in coronary artery disease is suggested by the observation that the extent of immunoreactive staining for ET-1 in atheromatous lesions is related to angina class.In line with this observation, ETA/ETB receptor blockade causes vasodilation, at least in certain patients with coronary atherosclerosis.


Restenosis is a major limitation of balloon angioplasty. Experimentally, the ET system is activated after vascular injury for several weeks.The extent of restenosis can be augmented by concomitant infusion of ET-1. Consequently, ET receptor blockade is effective in reducing neointima formation after balloon angioplasty in both rodents and pigs.

Pulmonary Hypertension

ET-1 expression in pulmonary tissue is increased in patients with primary and secondary pulmonary hypertension.Circulating ET-1 increases at high altitudes in mountaineers and correlates with pulmonary pressures and oxygen tension.ET increases even more in mountaineers prone to high-altitude pulmonary edema.Similar observations were made in patients with congestive heart failure.In heart failure, elevated ET-1 plasma levels are, at least in part, related to impaired ETB receptor–mediated clearance. Acute and short-term treatment of heart failure patients with the nonselective ET antagonist bosentan markedly lowers pulmonary artery pressure.However, the increase in circulating ET-1 during therapy suggests that ETB-mediated clearance is reduced by bosentan. In experimental studies of hypoxia-induced and monocrotaline-induced pulmonary hypertension, chronic ET receptor blockade lowered pulmonary artery pressures and the incidence of vascular and pulmonary injury and improved NO-mediated pulmonary vasodilatation. Similar observations were made in rats with high altitude–sensitive pulmonary hypertension.

Congestive Heart Failure and Left Ventricular Dysfunction

Heart failure due to coronary artery disease or hypertension is a major cause of morbidity and mortality. Although ACE-inhibitors, ß-blockers, and spironolactone reduce cardiovascular events, prognosis remains poor. In experimental animals and in patients with heart failure, the plasma levels of ET-1 are increased,and they predict survival.
The growth-promoting effects of ET-1 on cardiomyocytes have been implicated in the development of left ventricular hypertrophy. Cardiac growth can be augmented by hypoxia, which may be important in chronic ischemia. In addition, ET-1–mediated cardiac hypertrophy is enhanced by the renin-angiotensin system.However, the mechanism by which ET-1 affects the progression of left ventricular hypertrophy into heart failure seems to be biphasic. Prolonged exercise in rats leads to the upregulation of myocardial ET expression.Similarly, in early stages of heart failure, ET-1 maintains cardiac function as ETA receptor inhibition worsens contractility. ETA receptors, ECE, and the preproendothelin gene are upregulated in heart failure in rats and humans and in hamsters with dilated cardiomyopathy,and they contribute to impaired ventricular function.
In animal models of chronic heart failure, prolonged ET blockade improves cardiac hemodynamics, reduces ventricular dilatation, and prolongs survival .The time point for the initiation of treatment, however, may be important because ET blockade can interfere with scar formation in injured myocardium. Whether selective ETA or nonselective ET blockade should be favored in heart failure is unclear. Beneficial hemodynamic and clinical effects occur with ETA receptor blockade, both with selective and nonselective ET antagonists. However, concomitant ETB blockade markedly increases circulating ET-1 levels. Whether this is of clinical relevance is unknown. Endothelin antagonists increase blood flow in the forearm conduit arteries and skin microcirculation, an effect that seems to involve the release of NO mediated by the blockade of ETA receptors. Although increased ETB-mediated systemic vasoconstriction has been reported in patients with heart failure,ETB receptor blockade may abrogate the beneficial effects of ETA receptor blockade on cardiac hemodynamics and renal function in humans and animals with heart failure.

The first hemodynamic studies of ETA blockade in humans have been promising. In patients with severe congestive heart failure, acute infusion of the nonselective antagonist bosentan increased cardiac output and reduced systemic and pulmonary resistance.Similar data have been obtained with the selective ETA receptor antagonists BQ-123 and LU135252 . The beneficial clinical and hemodynamic effects of the blockade persist, and the increase in cardiac index is even more pronounced after 2 weeks of chronic treatment with bosentan. The Research on Endothelin Antagonism in Chronic Heart failure (REACH-1) trial with bosentan was terminated early because of hepatic side effects. The results showed an early worsening (at 3 months) and a potential benefit at 6 months, with decreased symptoms and reduced progression of heart failure. Possibly, the high dosages used without up-titration in the first weeks of therapy worsened heart failure in some patients. Lower dosages of bosentan are now being evaluated in the Endothelin Antagonist Bosentan for Lowering Cardiac Events in Heart Failure (ENABLE) trial. Whether selective ETA blockade will improve clinical symptoms and outcome in heart failure is currently being investigated in several small trials.

Endothelins and Endothelin Receptor Antagonists

Therapeutic Targets

Therapeutic Considerations for a Novel Class of Cardiovascular Drugs


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