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Endothelin and endothelin antagonism: Roles in cardiovascular health and disease
Endothelin and endothelin antagonism: Roles in cardiovascular health and disease Praveen Tamirisa, MD, William H. Frishman, MD, and Anil Kumar, MD Bronx, N.Y.
Endothelin is a naturally occurring polypeptide substance with potent vasoconstrictive actions. It was originally described as endotensin or endothelialcontracting factor in 1985 by Hickey et al., 1 who reported on the finding of a potent stable vasoconstricting substance produced by cultured endothelial cells. Subsequently, Yanagisawa et al.2 isolated and purified the substance from the supernatant of cultured porcine aortic and endothelial cells and then went on to prepare its complementary deoxyribonucleic acid (cDNA). This substance was renamed endothelin. Endothelin is the most potent vasoconstrictor known to date. Its chemical structure is closely related to certain neurotoxins (sarafotoxins) produced by scorpions and the burrowing asp (Atractaspis engaddensis). 3 Endothelins have now been isolated in various cell lines from several organisms. They are now considered to be autocoids or cytokines 4 because of their wide distribution, their expression during ontogeny and adult life, their primary role as intracellular factors, and the complexity of their biologic effects. In this review we discuss the biologic effects of endothelin and their receptors, the possible relation of endothelin to various pathophysiologic states, the role of endothelin as a disease marker, and the possible use of endothelin receptor blockade to treat various systemic illnesses. STRUCTURE
The superfamily of endothelins and sarafotoxins have two main branches with four members each. Endothelin is a polypeptide consisting of 21 amino
From the Department of Medicine, Division of Cardiology, The Albert Einstein College of Medicine-Montefiore Medical Center. Received for publication Nov. 30, 1994; accepted Jan. 10, 1995. Reprint requests: William H. Frishman, MD, Einstein College Hospital/ Montefiore Medical Center, 1825 Eastchester Rd., Bronx, NY 10461. AM HEARTJ 1995;130:601-10 Copyright © 1995 by Mosby-Year Book, Inc. 0002-8703/95/$3.00 + 0 4/1165063
acids. There are three closely related isoforms endothelin-1, endothelin-2, and endothelin-3 (ET1, ET2, and ET3, respectively), which differ in a few of the amino acid constituents (Fig. 1). The fourth member, called ET4 or vasoactive intestinal constrictor, is considered to be the murine form ofET2. 5 The endothelin molecules have several conserved amino acids, including the last six carboxyl (C)-terminal amino acids and four cysteine residues, which form two intrachain disulfide bonds between residues 1 and 15 and 3 and 11. These residues may have biologic implications particularly in relation to threedimensional structure and function. The main differences in the endothelin isopeptides reside in their amino (N)-terminal segments. There is a very high degree of sequence similarity between the two branches (approximately 60%) and within the constituent members of a branch (71% to 95%). PRODUCTION
Endothelin has been demonstrated to be produced from endothelial and nonendothelial cells. Factors known to release endothelin are shown in Table I. The synthesis of endothelins parallels that of the various peptide hormones in that a precursor polypeptide is sequentially cleaved to generate the active form (Fig. 2). Recently, endothelin-converting enzyme (ECE) was cloned. 5a ECE acts at an essential step in the production of active forms of endothelins. The fully formed molecule is then broken down into inactive peptides by as yet uncharacterized proteases. Some candidates are the lysosomal protective protein (deamidase) and enkephalinase (neutral endopeptidase EC 24.11). 6-s The regulation ofendothelin production occurs predominantly at the levels of transcription and translation. No storage vesicles containing endothelin have been identified. 9 The genes for the various endothelin isoforms have been sequenced and are found to be scattered in different chromosomes.Z° Current evidence suggests that they arose from a common ancestor by exon duplication 601
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Tamir~a, F/~h/?~n, and Kb~Tr/~r Table
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I.
Factors known to release endothelin
Thrombin Transforming growth factor-~ Arginine vasopressin Hypoxia Phorbol ester Glucose Angiotensin II Cyclosporin Insulinlike growth factor Bombesin Cortisol Low-density lipeprotein cholesterol Hypercholesterolemia Changes in shear stress on vascular wall
Table ]]. R e c e p t o r affinities
Receptor ETA ETB ETC
~CO0S6b CO0-
Fig. 1. Amino acid sequences ofET1, ET2, vasoactive intestinal constrictor (VIC) (possibly mouse or rat analogue of ET2), and ET3 as well as sarafotoxin S6b, one of the peptide constituents of venom of burrowing asp Atractaspis engadclensis, termed sarafotoxins, which, with other members of this peptide family, have remarkable homology to endothelins. (From Kramer et al. Circulation 1992; 85:351.) followed by gene duplication with subsequent dispersion of the different genes. On thechromosome upstream to the genes, regions such as the AP1 motif, 11 nuclear factor-binding element, 12 and acutephase reactant regulatory elements have been identified and contain the motifs that interact with transcription factors and serve as potential points for gene control and modulation. ENDOTHELIN RECEPTORS
The receptors for endothelin have been isolated and their genes cloned. 13-15The receptors are classified on the basis of their affinity for the various endothelin isoforms. So far two receptors, called ETA and ETB, have been well characterized. The gene for a third receptor could not be isolated by hybridiza-
Affinity ET1 > ET2 > ET3 ET1 = ET2 = ET3 ET3 > ET1
tion techniques with the cDNA of ETB, suggesting that it is less homologous to the other two receptors. In h u m a n beings ETA and ETB receptors exhibit significant sequence similarity, with about 63% amino acid identityJ 6 Evidence also points to the presence of subtypes among the various receptors.17, 18 The receptors appear to be glycoproteins; the sugar moiety is a possible important constituent for ligand interactions. 14 The ETA receptors probably recognize the tertiary structure of both the N-terminal and the C-terminal segments, whereas the ETB receptors recognize predominantly the Cterminal parts, explaining the differences in affinities of the two receptors for the isoforms (Table II). 9 The endothelin receptors belong to the rhodopsin superfamily, which also includes the B1, B2, 5HT1, 5HT2, V1, and V2 receptors. Members of this family are characterized by the presence of seven transmembrane segments in the receptor that span the membrane; their action is mediated by G proteins. There are many types of G proteins: inhibitory, stimulatory, and others. The binding of the ligand to the receptor induces an allosteric effect that causes corresponding changes in the interaction with the G proteins. It is thought that the third intracellular loop of the receptor is the site responsible for interacting with the G proteins. The exact residues of the receptor that interact with the ligand are not clearly known, and mutational studies are in progress to identify the responsible residues.
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Fig. 2. Pr~te~yticpr~cessing~fprepr~end~the~inandproendothelininbiosynthesisofmatureET1.From Highsmith HF et al. (Reproduced, with permission, from the Annual Review of Physiology, Volume 54, pages 257-77, © 1992 by Annual Reviews Inc.) The differential distribution of the endothelin receptors in various tissues is responsible for the multiplicity of actions attributed to endothelin (Table III). The binding of endothelin to its receptor is very tight, with relatively slow dissociation, which may result in prolonged action. An important factor to consider is that some of the effects seen in vitro may not be biologically relevant to events in vivo. 19 ETA receptors have been found predominantly in the heart, blood vessels of the brain and vascular smooth muscle, whereas the ETB receptors are more widely distributed and predominate in the kidney, uterus, central nervous system, and endothelial cells. MECHANISM OF ACTION
The binding of endothelin to its receptor has several effects (Fig. 3). Activation of phospholipase C causes hydrolysis of phosphatidylinositol, forming inositol-l,4,5-trisphosphate (IP3) and diacylglycerol. The newly formed IP3 causes release of calcium from the sarcoplasmic reticulum storage sites. It also facilitates the entry of extracellular calcium by dihydropyridine-sensitive voltage channels. The total intracellular free calcium concentration increases; the initial increase reflects the release from cellular stores, and the latter increase reflects net inward calcium entry across the cell membrane. 2°-23 The greater concentration of intracellular calcium often associates with calmodulin and modulates diverse processes such as neurotransmitter release, secretion, and activation of enzymes including myosin light-chain kinases that activate cellular contractile machinery and other mechanisms. 24 Nifedipine and
Fig. 3. Intracellular signal transduction pathways activated by endothelins (ETs). Activated ET receptor stimulates phospholipase C (PLC) and phospholipase A2 (PLA2). Activated ET receptor also stimulates voltage-dependent calcium channels (VDC) and probably receptor-operated calcium channel (ROC). Inositol triphosphate (IP3) elicits release of calcium ion from caffeine-sensitive calcium store. Protein kinase C (PKC) activated by diacylglycerol (DG) sensitizes contractile apparatus. Increased concentration of intracellular free calcium ion ([Ca2+]~induces contraction. Cyclooxygenase products (prostacyclin [PGI2], prostaglandin E2 [PGE2], and thromboxane A2 [TXA2]) modify contraction. G, G protein; IP2, inositol biphosphate; IP3, inositol triphosphate; PIP2, phosphatidyl inositol biphosphate. (From Masaki T et al. Circulation 1991;84: 1460.)
other calcium-channel blocking agents can inhibit the influx of extracellular calcium, thereby antagonizing some of the actions of endothelin. 25-27 The sustained release of DAG causes prolonged
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604 Tamirisa, Frishman, and Kumar Table III. Two distinct pharmacologic activities of endothelins Receptor affinity rank order
System 1 (ETAtype): ET1 > ET3
Tissue or organ and species
Vascular smooth muscle cells Airway smooth muscle (human) Uterine smooth muscle (rat) Cardiac muscle (neonatal rat) Adrenocorticalcells (zona glomerulosa, bovine) C6 glioma cells (rat) Fibroblast cells (human, mouse) Osteoblast cells (rat)
System 2 (ETBtype) ET1 = ET3
Vascular smooth muscle cells (rat) Mesenteric artery (rat) Renal pelvis smooth muscle (human) Stomach (rat) Blood (rabbit) Cerebral cortex,cerebellum(rat) Adrenocorticalcells (bovine) Astrocyte (rat) Mesangium cells (rat)
Assay
Pressor effectin vivo, contraction,PI generation, binding assay Contraction Contraction, PI generation, binding assay PI generation, binding assay Stimulation of aldosterone secretion, binding assay PI generation, binding assay Calcium transient,* binding assay DNA synthesis, PI generation, binding assay Initial depressor response Transient relaxation, NO release Contraction Ulcerogenicity Inhibition of platelet aggregation ex vivo PI generation Binding assay PI generation, calcium transient PI generation,binding assay
Endothelin-induced responses may be divided into two groups according to pharmacologic Potency Rank order among the three endothelin isopeptides.
DNA, Deoxyribonucleic acid; NO, nitric oxide (enothelium-derived relaxing factor) PI, phosphoinositide (inositol phosphate). *Transient increase in intracellular calcium evoked by endothelins. (Adapted from Sakurai T, Goto IL Drugs 1993;46:795-804.)
activation of protein kinase C (PKC), 28 which, by causing phosphorylation of various proteins, m ay result in activation or inactivation of proteins and the regulation of gene expression. Endothelin has also been noted to cause increase in intracellular pH, perhaps by stimulating the sarcolemmal sodium ion-hydrogen ion antiporter. This increased intracellular pH can enhance the sensitivity of the myofilaments to calcium, thereby augmenting contraction even without changing the calcium concentration.29, 30 It has also been noted t h a t the increase in intracellular pH can by itself stimulate hypertrophic and mitogenic responses in different cells. 31, 32 The binding of endothelin to its receptor also m ay alter ion-channel permeability (such as t ha t of the voltage-dependent L-type calcium channels by G proteins) and cause activation of other secondary messengers (such as production of prostanoids by activating phospholipase A)33 and release of nitric oxide. 34-36Nitric oxide has been shown to antagonize some of the effects ofendothelin on vascular smoothmuscle contraction, perhaps by decreasing endothelin release from endothelial cells. 37 In addition, experiments have revealed a competitive inhibition of ET1 binding to its receptors in cardiac membranes of rats by potassium-channel opening agents. These agents have been shown to exert cardioprotective
action after experimental myocardial infarction in rats by suppressing cardiac arrhythmias, which may result in part from inhibition of the effects of cardiac endothelin. It has been suggested t hat ET1 m a y close adenosine triphosphate-sensitive potassium channels.38-40 PATHOPHYSIOLOGIC ROLES OF ENDOTHELIN Systemic hypertension. Endothelin is the most potent vasoconstrictor known to date and has an exceptionally long duration of physiologic action. 9 The influence of endothelin in maintaining normal blood pressure and its role in the cause of systemic hypertension remain unclear. 41 Intravenous injections of endothelin in animals cause a transient decrease in systolic blood pressure (ETB) followed by a prolonged pressor response (ETA). 2, 11 The vasoconstrictor action is mediated by ETA receptors in the vascular smooth muscle, whereas the predominant vasodilation effect is mediated by the ETB receptors on the endothelial cells t h a t cause release ofprostacyclin and nitric oxide. Therefore the overall predominant hemodynamic effect of endothelin in a given organ depends on the receptor type being stimulated, its location, and its relative abundance. Angiotensin II has been found to increase endothelin concentrations (Table IV) in vitro from endo-
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thelial cells, suggesting one mechanism by which angiotensin-converting-enzyme (ACE) inhibition could function in vivo. 42 ACE inhibitors also can indirectly interfere with endothelin: increased concentrations of bradykinin decrease endothelin release (by acting through bradykinin 2 receptors, stimulation of which cause increased nitric oxide release). ACE inhibitors can cause regression of intimal hyperplasia, whereas other antihypertensive drugs are ineffective in this regard. 43 The significance of plasma endothelin concentrations in hypertension has been subject to controversy. Endothelin has been implicated as the cause for cyclosporine-induced hypertension. After the development of new specific and sensitive assays for endothelin, it was shown that there were no significant differences in the endothelin concentrations of patients with normal blood pressure and with hypertension. 44 The previously reported higher plasma concentrations ofimmtmoreactive endothelin in subjects with hypertension is now attributed to cross reactivity of the older assays. On the other hand, an increased sensitivity to available endothelin has been reported in some studies. 45, 46 The plasma concentrations of endothelin tend to increase with age and are higher in men than women in both normotensive and hypertensive subjects. 44 Atherosclerosis. Hypercholesterolemia leads to profound alterations in the regulation of vascular tone. Low-density lipoprotein accumulates in vascular walls in hypercholesterolemia. Experimental studies have shown that oxidatively modified low-density lipoproteins induce the production of ET1 by h u m a n macrophages and can increase endothelin formation and release from endothelial cells. 47, 4s High plasma concentrations of endothelin have been described in patients with atherosclerosis. 49 One of the early mechanisms of atherosclerosis includes the migration of monocytes into the intimal layer of blood vessels, where they become activated and stimulate the release of multiple factors. It has been demonstrated in vitro that endothelin can serve as a chemotactic agent for monocytes, and therefore its role as an initiator of the atherosclerotic process has been strengthened. Endothelin also may stimulate the migration and proliferation of vascular smooth-muscle cells, t h e r e b y sustaining the atherosclerotic p r o c e s s . 27, 50, 51 Calcium-channel blocking agents have been shown to reduce the chemotatic movements of monocytes, perhaps by interfering with calcium ion fluxes. Myocardial ischemia. Myocardial ischemia can enhance the release of endothelin by cardiomyocytes and increase its vasoactive effects. Infusion of the
Tamirisa, Frishman, and Kumar 605 Table iV. Conditions associated with increased circulating
endothelin in blood Acute myocardial infarction Allograi~ rejection Atherosclerosis Cold exposure Coronary spasm Cardiomyopathy Disseminated intravascular coagulation Diabetes mellitus Exercise Essential hypertension Hemodialysis Hypertension of pregnancy Ischemic heart disease Preeclampsia Pregnancy Pulmonary hypertension Postural changes Raynaud's phenomenon Stable chronic renal failure Transplantation of organs Trauma Uremia Subarachnoid hemorrhage Surgery Sepsis Scleroderma Shock
ET1 isoform directly into the coronary circulation of animals results in the development of myocardial infarction, with impaired ventricular functioning and the development of arrhythmias. 52 Endothelin has been shown to lower the threshold for ventricular fibrillation in dogs. 53 An increase in ET1 has been observed in cardiac tissue after experimental myocardial infarction in rats, and pretreatment with an antiendothelin ~-globulin in this model can reduce infarct size by as much a s 40%. 54 Infusion of ETAreceptor antagonist drugs before an ischemic insult can also reduce infarct size in animals. 55 Plasma endothelin concentrations and urinary endothelin excretion are increased in patients with myocardial infarction 56 and in vasospastic angina but not in stable or unstable angina. 51, 54, 57 Patients who undergo successful thrombolysis with early reperfusion have lower endothelin concentrations than subjects who do not have early reperfusion. Plasma endothelin concentrations can also predict hemodynamic complications in patients with myocardial infarction. 5s Patients with the highest plasma endothelin concentrations after myocardial infarction have the highest creatine phosphokinase (CPK) and CPK MB-isoenzyme concentrations and the lowest angiographically determined ejection fractions. 59 It has also been suggested that the serum
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concentration of endothelin can be used as a prognostic indicator of survival after acute myocardial infarction. Left ventricular function and congestive heart failure.
Endothelin exhibits potent inotropic activity in isolated hearts, cardiac muscle strips, isolated cells, and instrumented intact animals. 6° High-affinity receptors for endothelin have been demonstrated in the atria and the ventricles. 61-63 Intravenous administration of the ET1 isoform produces delayed prolonged augmentation of left ventricular performance in addition to its biphasic vasoactive effects of transient vasodilation followed by sustained vasocontraction. 60 Endothelin is a potent secretogne of atrial natriuretic factor, which is a naturally occurring antagonist of endothelin. 64 The ETA receptor appears to mediate endothelin's actions of vasoconstriction and the stimulation of atrial natriuretic factor secretion, and the ETB receptor mediates endothelin-induced vasodilation and activation of the renin-angiotensin-aldosterone system. Urinary water excretion is mediated through both receptors, but sodium excretion is mediated through the ETA receptor. Increased concentrations of endothelin described in patients with congestive heart failure 65, 66 are predictive of increased mortality r i s k . 66 It also has been suggested that increased concentrations of endothelin may play an important role in the increased systemic vascular resistance observed in congestive heart failure. 65 Increased endothelin concentrations have also been observed in the plasma and hearts of cardiomyopathic Syrian hamsters 67 and in the cells of endothelial cells infected with Trypanosoma cruzi in experimental Chagas' cardiomyopathy. 6s There is early clinical evidence that treatment with ETA receptor antagonists and ECE inhibitors can influence favorably the course of h u m a n heart failure. 69 ACE inhibitors may also benefit patients with heart failure because of their antiendothelin actions. 7° Pulmonary hypertension. Expression of ET1 in the lung has been studied 7°a by immunocytochemistry and hybridization in situ in specimens from patients with pulmonary hypertension of primary or secondary causes. In contrast to normal lung, specimens from patients with pulmonary hypertension exhibit abundant ET2 immunostaining, particularly over endothelium of markedly hypertrophied muscular pulmonary arteries and plexogenic lesions. Endothelin has been suggested as a potent vasoconstrictor and growth-promoting factor in the pathophysi-
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ologic mechanisms of pulmonary hypertension. 71-74 In animals, an orally active ETA and ETB receptor blocking agent, bosentan, has been shown to prevent hypoxia-induced pulmonary hypertension and pulmonary artery remodeling. 72, 75 Endothelin has been proposed to be a local modulator for oxygen to cause closure of the ductus arteriosus after birth. 76 Increased endothelin concentrations are found in various congenital heart disorders associated with left-to-right shunts. 77 Ventricular and vascular hypertrophy. Endothelin increases DNA synthesis in vascular smooth-muscle ceils,7S, 79 cardiomyocytes,80 fibroblasts, glial cells, mesangial cells, and other cells; causes expression of protooncogenes; causes cell proliferation27; and causes hypertrophy, sl It acts in synergy with various factors such as transforming growth factor, epiderreal growth factor, platelet-derived growth factor, basic fibroblast growth factor and insulin to potentiate cellular transformation and replication. This synergy suggests that all of these factors act through common pathways involving PKC and cyclic adenosine monophosphate. Endothelin per se may not be a direct mediator of angiogenesis but may function as a comitogenic factor. 27 The role of endothelin as a mitogen for vascular smooth-muscle cells is unclear. It has been found that externally added endothelin to the cultured rat aortic smooth-muscle cell does not induce proliferation, but when the same cells are transfected with endothelin producing plasmids, cell lines that produce the greatest amount of endothelin show the greatest proliferation, suggesting an autocrine action. In addition, this effect is significantly reduced by ETA-specific antagonists. 82, s3 Increased protein synthesis induced by endothelin may play a role in myocardial hypertrophy, s4,s5 Marked increase in prepro-ET1 messenger ribonucleic acid and mature ET1 has been found in the ventricles of rats in which concentric hypertrophy was induced experimentally, s6, s7 Regulation ofgene expression as shown by increased levels of c-fos, c-myc, fra 1, jun b, and other genes has been found in cells stimulated by endothelin, s°, s5, 88 The regnlation of ET1 gene transcription by fos andjun oncogenes, 89 which in turn are regulated by ET1, creates a loop, which makes matters more complex, Neointima formation after vascular wall trauma. The efficacy of coronary angioplasty is limited by the high incidence ofrestenosis. ET1 induces cultured vascular smooth-muscle cell proliferation by activation of the ETA-receptor subtype, a response that normally
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is attenuated by an intact, functional endothelium. In addition, ET1 also induces the expression and release of several protooncogenes and growth factors that modulate smooth-muscle cell migration, proliferation, and matrix formulation. In addition to inhibiting smooth-muscle cell proliferation in vitro, endothelin-receptor antagonism with SB 209670 ameliorates the degree of neointima formation observed after rat carotid artery angioplasty. The observations raise the possibility that ET1 antagonists will serve as novel therapeutic agents in the control of restenosis. 83 AIIograft rejection. ET1 concentrations are increased in patients with graft rejection and arteriosclerosis. This increase in endothelin activity may contribute to altered microvascular hemodynamics and remodeling and to the long-term hypertension and nephrotoxicity seen in patients undergoing transplantation. 90 Cerebrovascular disease. Laboratory and clinical investigations suggest that endothelin play a role in the pathogenesis of delayed cerebral vasospasm in subarachnoid hemorrhage and ischemic stroke. High concentrations of endothelin have been demonstrated in the cerebral spinal fluid of these patients. 91 Endothelin-receptor inhibitors have been suggested as treatments for patients with subarachnoid hemorrhage to influence cerebral vasoconstriction and delaying neuronal death. Renal function, acute renal failure, and nephrotoxicity. Endothelin can cause increased renal vascular resistance, decreased renal blood flow, and decreased glomerular filtration, effects that resemble those seen in renal failure. In some studies, the infusion of antiendothelin antibodies has been found to exert a renal protective effect in ischemia 9294 and in cyclosporine nephrotoxicity. 95 However, in a recent study, the use of a specific ETA receptor blocking agent was found not to reverse chronic cyclosporine-induced vasoconstriction. 96 ENDOTHELIN ANTAGONISTS
Active research and drug development programs are aimed at investigating endothelin inhibitors. Preventing the possible pathophysiologic effects of endothelin at their receptors with selective antagonists is a new frontier in pharmacotherapy. Several antagonists to endothelin have been discovered in recent years (Table V). The original receptor antagonists were isolated from the cultured broth of Streptomyces misakiensis. Some of the agents are receptor specific for the ETA or the ETB subtype, but
Tamirisa, Frishman, and Kumar 607 Table V. Endothelin receptor antagonists ETA
ETB
Nonspecific
BQ 123 BQ 153 FR 139317 Asterric acid BMS 182874
IRL 1038 BQ 788 RES 701-1
PD 145065 PD 142893 Cchinmicins I, II, and III Ro 462005 Bosentan SB 209670 Phosphoramidon (ECE inhibitor)
Table VI. Nonspecific endothelin antagonists ECE inhibitors Angiotensin-converting-enzyme inhibitors Angiotensin II receptor blocking agents Calcium-entry blocking agents Potassium-channel opening agents Adenosine Nitroglycerin These antagonists interfere with release or modify action of endothelin.
others are non-specific. There also are other classes of drugs (Table VI) that interfere with endothelin release or modify its actions. Efforts are now underway to customize specific antagonists, and according to the known structure of endothelin, to modify key amino acids to create innovative compounds. Another innovative approach is to control endothelin action by interfering with the ECE essential for the production of the active compounds. The use of endothelin antagonists that can be used intravenously or orally will help to elucidate the mechanisms by which endothelin mediates its effects. These drugs m a y also be of use in the treatment of patients with systemic hypertension, myocardial ischemia, restenosis after angioplasty, congestive heart failure, subarachnoid hemorrhage, renal failure (radiocontrast induced), allograft rejection, or sepsis. Clinical trials of the use of endothelin antagonists for many of these conditions are now in progress. SUMMARY
Endothelin is the most potent mammalian vasoconstrictor yet discovered. Its three isoforms play leading roles in regulating vascular tone and causing mitogenesis. The isoforms bind to two major receptor subtypes (ETA and ETB), which mediate a wide variety of physiologic actions in several organ systems. Endothelin may also be a disease marker or an etiologic factor in ischemic heart disease, atheroscler0-
,
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sis, congestive heart failure, renal failure, myocardial and vascular wall hypertrophy, systemic hypertension, pulmonary hypertension, and subarachnoid hemorrhage. Specific and nonspecific receptor antagonists and ECE inhibitors that have been developed interfere with endothelin's function. Many available cardiovascular therapeutic agents, such as angiotensin-converting-enzyme inl~ibitors, calciumentry blocking drugs, and nitroglycerin, also may interfere with endothelin release or may modify its activity. The endothelin antagonists have great potential as agents for use in the treatment of a wide spectrum of disease entities and as biologic probes for understanding the actions of endothelin in human beings. REFERENCES
1. Hickey KA, Rubanyi GM, Paul RJ, Highsmith RF. Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am J Physiol 1985;248:550-6. 2. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-5. 3. Kloog Y, Ambar I, Sokolovsky M, Kochva E, Wollberg Z, Bdolah A. Sarafotexin, a novel vasoconstrictor peptide: phosphoinositide hydrolysis in rat heart and brain. Science 1988;242:268-70. 4, Kramer BK, Nishida M, Kelly RA, Smith TW. Endothelins: myocardial actions of a new class of cytokines. Circulation 1992;85:350-4. 5. Bloch DK, Hong CC, Eddy RL, Shows TB, Quertermous T. cDNA cloning and chromosomal assignment of the endothelin-2 gene: vasoactive intestinal contractor peptide is rat endothelin-2. Genomics 1991; 10:236-42. 5a. Shimada K, Matsushita Y, Wakabayashi K, Takahashi M, Matsubara A, Lijima ¥, Tanzawak t~ Cloning and functional expression of h u m a n endothelin converting enzyme CDNA. Biochem Biophys Res Commun 1995;207:807-12. 6. Jackman KL, Morris PW, Rabito SF, Johansson GB, Skidgel RA, Erdos EG. Inactivation of endothelin 1 by an enzyme of the vascular endothelial cells. Hypertension 1993;21:925-8. 7. Sokolovsky M, Galron R, K]oog Y, Bdolah A, Indig FE, Blumberg S, Fleminger G. Endothelins are more sensitive than sarafotoxins to neutral endoproteinase: possible physiological significance. Proc Natl Acad Sci U S A 1990;87:4702-6. 8. Vijayaraghavan J, Scicli AG, Carretero OA, Slaughter C, Moomaw C, Hersh LB. The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase). J Biol Chem 1990;265:14150-5. 9. gakural T, Goto K. Endothelins: vascular actions and clinical implications. Drugs 1993;46:795-804. 10. Yanagisawa M, Masaki T. Molecular biology and biochemistry of the endothelins. Trends Pharmacol Sci 1989;10:374-8. 11. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The h u m a n endothelin family: three structurally and pharmacologicaUy distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A 1989;86:2863-7. 12. MiyauchiT,Yanagisawa M, TomJzawa T, Sugishita Y, Suzuki N, Jujino M, Ajisaka R, Goto K, Masaki T. Increased plasma concentrations of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet 1989;2:53-4. 13. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a c D N A encoding an endothelialreceptor.Nature 1990; 348:730-2. 14. Lin HY, Kaji EH, Winkel GK, Ives HE, Lodish HF. Cloning and expression of a vascular smooth endothelin I receptor. Proc Natl Acad Sci U S A 1991;88:3185-9. 15.. Sakurai T, Yanagisawa M, Masaki T. Molecular characterisation of the endothelin receptors. Trends Pharmacol Sci 1992;13:103-8.
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16. Sakamoto A, Yanagisawa M, Sakurai T, Takuwa Y, Yanagisawa H, Masaki T. Cloning and functional expression of h u m a n cDNA for ETB endothelin receptor. Biochem Biophys Res Commun 1991;178:656-63. 17. Cristol JP, Warner TD, Thiemermann C, Vane JR. Mediation via different receptors of the vasoconstrictor effects of endothelins and sarafotoxins in the systemic circulation and renal vasculature of the anesthetized rat. Br J Pharmacol 1993;108:776-9. 18. Sudjarwo SA, Hori M, T~kai M, Urade Y, Okada T, Karaki H. A novel subtype of endothelin B receptor mediating contraction in swine pulmonary vein. Life Sci 1993;53:431-7. 19. Nathan C, Sporn M. Cytokines in context. J Cell Biol 1991;113:981-6. 20. Chan J, Greenberg DA. Endothelin and calcinm signalingin NG 108-15 neuroblastoma X glioma cells. J Pharmacol Exp Ther 1991;258:524-30. 21. Ohnishi A, Yamaguchi K, Kusuhara M, Abe K, Kimura S. Mobilization ofintracellular calcium by ET in Swiss 3T3 cells. Biochem Biophys Res Commun 1989;161:489-95. 22. Takuwa N, Takuwa Y, Yanagisawa M, Yamashita K, Masaki T. A novel vasoactive peptide stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Biol Chem 1989;264:7856-61. 23. Takuwa Y, Masaki T, Yamashita K. The effects of the endothelin family peptides on cultured osteoblastic cells from rat calvariae. Biochem Biophy Res Commun 1990;170:998-1005. 24. Miller RC, Pelton JT, Huggins JP. Endothelins: from receptors to medicine. Trends Pharmacol Sci 1993;14:54-60. 25. Nakaki T, Nakayama M, Yamamoto S, Kato R. Endothelin-mediated stimulation of DNA synthesis in vascular smooth muscle cells. Biochem Biophy Res Commun 1989;158:880-3. 26. Nilsson J, Sjolund M, Palmberg L, Von Euler AM, Jonzon B, Thyberg J. The calcium antagonist nifedipine inhibits arterial smooth muscle proliferation. Atherosclerosis 1985;58:109-22. 27. Battistini B, Chailler P, D'Orleans-Juste P, Briere N, Sirois P. Growth regulatory properties of endothelins. Peptides 1993;14:385-99. 28. Brown KD, Littlewood CJ. Endothelin stimulates DNA synthesis in Swiss 3T3 cells, synergy with polypeptide growth factors. Biochem J 1989;263:977-80. 29. Fabiato A, Fabiato F. Effects of pH on the myofilaments and the sarcoplasmic reticalum of skinned cells from cardiac and skeletal muscles. J Physiol (Land) 1978;276:233-55. 30. Allen DG, Orchard CH. The effects of changes of pH on intra-cellular calcium transits in mammalian cardiac muscle. J Physiol (Lond) 1983; 335:555-67. 31. Komuro I, Kurihara H, Sugiyama T, Takaku F, Yazaki Y. Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988;238:249-52. 32. Simonson MS, Wann S, Mene P, Dubyak GR, Kester M, Nakazate Y, Sedor JR, Dunn MJ. Endothelin stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells. J Clin Invest 1989;83:708-12. 33. Chakravarthy U, Archer DB. Endothelin: a new vasoactive ocular peptide. Br J Ophthalmol 1992;76:107-8. 34. DeNucci G, Thomas R, D'Orleans-Juste P, Antunes E, Walker C, Warner TD, Vane JR. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelin derived relaxing factor. Proc Natl Acad Sci U S A 1988;85:9797-800. 35. Nakami A, Hirata Y, Isbikawa M, Moroi M, Aikawa J, Mashii K. ET-1 and ET-3 induce vasorelaxation via common generation ofendothdinm derived nitric oxide. Life Sci 1992;50:677-82. 36. Nakamuta M, Takayanagi R, Sakai Y, Sakamoto S, Hagiwara H, Mijuno T, Saito Y, Hirosa S, Yamamoto M, Nawata H. Cloning and sequence analysis of a cDNA encoding h u m a n non-selective type of endothelin receptor. Biochem Biophys Res Commun 1991;177:34-9. 37. Vincent R, Hogie M, Clozel M, Thuillez C. In vivo evidence of an endothelin-induced vasopressor tone after inhibition of nitric oxide synthesis in rats [Abstract]. Circulation 1994;90:I-35. 38. Haynes WG, Waugh CJ, Dockrell MEC, Olverman HJ, Williams BC, Webb DJ. Modulators of calcium and potassium channels: their effects on endothelin-1 binding to cardiac membranes. J Cardiovasc Pharmacol 1993;22(suppl):S154-7. 39. Grover GJ, Dzwonczyk S, Parham CS, Sleph PG. The protective effects of cromakalim and pinacidil on reperfusion function and infarct size in anesthetized dogs. Cardiovasc Drugs Ther 1990;4:465-74.
Volume 130, Number 3, Part 1 American Heart Journal
40. Kerr MJ, Wilson R, Shanks RG. Suppression of ventricular arrhythmias after coronary artery ligation by pinacidil, a vasodilator drug. J Cardiovasc Pharmacol 1985;7:875-83. 41. Donckier J, Stoleru L, Hayashida W, Van Mechelen H, Galanti L, Ketelslegers J-M, Pouleur H. Role of endogenous endothelin-1 in experimental hypertension [Abstract]. Circulation 1994;90:I-387. 42. Ciafre SA, D'Armiento FP, DiGregorio F, Colasanti P, DiBenedette A, Langella A, Di Ieso N, Liguori A, Colasanti R, Napoli C. Angiotensin II stimulates endothelin-1 release from h u m a n endothelial cells. Recenti Prog Med 1993;84:248-53. 43. Powell JS, Clozel JP, Muller RK, Kuhn H, Hefti F, Hosang M. Inhibitors of angiotensin converting enzyme prevent myointimal proliferation after vascular injury. Science 1989;245:186-8. 44. Miyauchi T, Yanagisawa M, Iida K, Ajisaka R, Suzuki N, Fujino M, Goto K, Masaki T, Sugishita Y. Age- and sex-related variation of plasma endothelin-1 concentration in normal and hypertensive subjects. AM HEARTJ 1992;123:1092-3. 45. Catelli DeCarvalho MH, Nigro D, Scivolette R, Barbeiro HV, Aparecida deOliveira M, deNucci G, Fortes ZB. Comparison of the effect of endothelin on microvessels and macrovessels in Goldblatt II and deoxycorticosterone acetate-salt hypertensive rats. Hypertension 1990;15(suppl I):I68-71. 46. Tomobe Y, Miyauchi T, Saito A, Yanagisawa M, Kimura S, Goto K, Masaki T. Effects ofendothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats. Eur J Pharmaco11988;152:373-4. 47. Boulanger CM, Tanner FC, Bea ML, Hahn AWA, Werner A, Luscher TF. Oxidized low density lipoprotein induces mRNA expression and release of ET from h u m a n and porcine endothelium. Circ Res 1992; 70:1191-7. 48. Martin-Nizard F, Houssaini HS, Lestavel-Delattre S, Duriez P, Fruehart JC. Modified low density lipoproteins activates macrophages to secrete ir-ET. FEBS Lett 1991;293:127-30. 49. Lerman A, Edwards BS, Hallett JW, Heublein DM, Sandberg SM, Burnett JC Jr. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis: N Engl J Med 1991;325:997-1001. 50. Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular diseases. Circ Res 1986;58:427-44. 51. Haynes WG, Webb DJ. The endothelin family: local hormones with diverse roles in health and disease? Clin Sci 1993;84:485-500. 52. Karwatowska-prokopezuk E, Wennmalm A. Effects of endothelin on coronary flow, mechanical performance, oxygen uptake and formation of purines and on outflow of prostacyclin in the isolated rabbit heart. Circ Res 1990;66:46-54. 53, Salvati P, Chierchia S, Dho L, Ferrario RG, Parenti P, Vicedomini G, Patrone C. Proarrhythmic activity ofintracoronary endothelir/in dogs: relation to the site of administration and to changes in regional flow. J Cardiovase Pharmacol 1991;17:1007-14. 54. Watanabe T, Suzuki N, Shimamoto N, Fujino M, Imada A. Contribution of endogenous endothelins to the extension of myocardial infarct size in rats. Circ Res 1991;69:370-7. 55. Grover GJ, Dzwonczyk S, Parham CS. The endothelin-1 receptor antagonist BQ-123 reduces infarct size in a canine model of coronary occlusion and reperfusion. Cardiovasc Res 2993;27:1613-8. 56. Omland T, Lie RT, Aakvaag A, Aarsland T, Dickstein K. Plasma endothelin determination as a prognostic indicator of i year mortality after acute myocardial infarction. Circulation 1994;89:1573-9. 57. Ray SG, McMurray JJ, Morton JJ, Dargie HJ. Circulating endothelin in acute ischemic syndromes. Br Heart J 1992;67:383-6. 58. Leehleitner P, Genser N, Mair J, Maier J, Artner-Dworzak E, Dienstl F, Puschendorf B. Endothelin-1 in patients with complicated and uncomplicated myocardial infarction. Clin Investig 1992;70:1070-2. 59. Lechleitner P, Genser N, Mair J, Maier J, Artner-Dworzak E, Dienstl F, PuschendorfB. Plasma immunoreactive endothelin in the acute and subacute phases of myocardial infarction in patients undergoing fibrinolysis. Clin Chem 1993;39:955-9. 60. Ohno M, Li W, Cheng C-P. Effects of endothelin-1 on lei~ ventricular performance in conscious dogs: assessment by pressure-volume analysis [Abstract]. Circulation 1994;90:I~16. 61. Gu X-H, Casley D, Nayler W. Specific high affinity binding sites for 125I-labelled porcine endothelin in rat cardiac membranes. Eur J Pharmacol 1989;167:281-90. 62. Galron R, Kloog Y, Bdolah A, Sokolovsky M. Functional endothelin/sa-
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rafotoxin receptors in rat heart myocytes: structure activity relationships and receptor subtypes. Biochem Biophys Res Commun 1989; 163:936-43. 63. Hirata Y. Endothelin-1 receptors in cultured vascular smooth muscle cells and cardiocytes of rats. J Cardiovasc Pharmacol 1989;13(suppl):S157-8. 64. Moe GW, Ferrazzi S, Naik G, Howard RJ. Endothelin in heart failure: Temporal evolution, source of production and interaction with atrial natrinretic peptide [Abstract]. Circulation 1994;90:I-592. 65. Teerlink JR, Hess P, Clozel M, Clozel J-P, Hoffman F. Role of endothelin in conscious rats with chronic heart failure [Abstract]. Circulation 1994;90:I-261. 66. Galatius-Jensen S, Wroblewski H, Emmeluth C, Bie P, Haunso S, Kastrup J. Plasma endothelin-1 in chronic heart failure: a predictor of cardiac death? Circulation 1994;90:I-379. 67. Inada T, Tanaka M, Hasegawa K, Ohtani S, Doyama K, Fujiwara T. Increased levels of endothelin-1 in plasma and heart tissue of cardiomyopathic Syrian hamsters [Abstract]. Circulation 1994;90:I-260. 68. Wittner M, Morris SA, Christ GJ, Hatcher VB, Zeballos GA, Bilezikian JP, Weiss LM, Tanowitz HB. ~nfection of cultured human endothelial cells increases endothelin levels [Abstract]. Circulation 1994;90:I-293. 69. Love MP, Haynes WG, Webb DJ, McMurray JJV. Anti-endothelin therapy is of potential benefit in heart failure [Abstract]. Circulation 1994;90:I-547. 70. Clavell AL, Mattingiy MM, Nir A, Aarhus LL, Heublein DM, Burnett JC Jr. Angiotensin converting enzyme inhibition modulates circulating and tissue endothelin activity in experimental heart failure [Abstract]. Circulation 1994;90:I-452. 70a. Stewart D. ET1 in pulmonary hypertension. Proceedings of the IBCsponsored meeting, Endothelin inhibitors: Advances in therapeutic application and development, June 9-10, 1994, Philadelphia, Pa. 71. Yamamoto K, Ikeda U, Mite H, Fujikawa H, Sekiguchi H. Endothelin production in pulmonary circulation of patients with mitral stenosis [Abstract]. Circulation 1994;90:I-172. 72. DiCarlo VS, Chen S-J, Chen Y-F, Yano M, Oparil S. Chronic blockade of endothelin-A receptors with BQ-123 prevents hypoxia induced pulmonary hypertension and pulmonary vascular remodeling in rats [Abstract]: Circulation 1994;90:I-6. 73. Stewart DJ, Levy RD, Cernacek P, Langieben D. Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease? Ann Intern Meal 1991;114:464-9. 74. Giaid A, Yanagisawa M, Langleben D, Michel R, Levy R, Shennib H, Kimura S, Masaki T, Duguid W, Stewart D. Expression ofendothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 1993;328:1732-9. 75. Chen S-J, Chen Y-F, Meng C, Oparil S. The endothelin receptor antagonist bosentan prevents short term hypoxia induced pulmonary hypertension in the rats [Abstract]. Circulation 1994;90:I-152. 76. Coceani F, Kelsey L, Seidlitz E. Evidence for an effector role of endothelin in closure of the ductus arteriosus at birth. Can J Physio] Pharmacol 1992;70:1061-4. 77. Vincent JA, Ross RD, Kassab J, Hsu JM, Pinsky WW. Relation of elevated plasma endothelin in congenital heart disease to increased pulmonary blood flow. Am J Cardiol 1993;71:1204-7. 78. Bunchman TE, Brookshire CA. Cyclosporine induced synthesis ofendothelin by cultured h u m a n endothelial cells. J Clin Invest 1991; 88:310-4. 79. Hassoun PM, Thappa V, Landman MJ, Fanburg BL. Endothelin-l: Mitogenic activity on pulmonary artery smooth muscle cells and release from hypoxic endothelial cells. Proc Soc Exp Biol Med 1992; 199:165-70. 80. Ire H, Hirata Y, Hiroe M, Tsujino M, Adachi S, Takamoto T, Nitta M, Taniguchi K, Marumo F. ET-1 induces hypertrophy with enhanced expression of muscle specific genes in cultured neonatal rat cardiomyocytes. Circ Res 1991;69:209-15. 81. Karam H, Heudes D, Hess P, Basset A, Clozel J-P, Clozel M. Role of endothelin in cardiac remodeling in DOCA hypertensive rats [Abstract]. Circulation 1994;90:I-261. 82. Alberts GF, Peifley KA, Johns A, Kleha JF, Winkles JA. Constitutive endothelin-1 overexpression promotes smooth muscle cell proliferation via an external autocrine loop. J Biol Chem 1994;269:10112-8. 83. Douglas SA, Louden C, Ohlstein Ett. Effect of acute and chronic
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administration of the endothelin receptor antagonists BQ-123 and SB 209670 on neointima formation following rat carotic artery balloon angioplasty [Abstract]. Circulation 1994;90:I-297. Chua BH, Kreba CJ, Chua CC, Diglio CA. Endothelin stimulates protein synthesis in smooth muscle cells. Am J Physiol 1992;262:E412-6. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, Chien KR. ET induction of inositel phospholipid hydrolysis, sarcomere assembly and cardiac gene expression in ventricular myocytes. J Biol Chem 1990;265:20555-62. Yorikane R, Sakai S, Miyauchi T, Sakurai T, Goto K. Possible involvement of endothelin-1 in cardiac hypertrophy. Arzneimittelforschung 1994;44:412=5. Yorikane R, Sakai S, Miyauchi T, Sakurai T, Sugishita Y, Goto K. Im creased production of endothelin-1 in the hypertrophied rat heart due to pressure overload. FEBS Lett 1993;332:31-4. Komuro I, Kurihara H, Sugiyama T, Takaku F, Yazaki Y. Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988;238:249-52. Lee ME, Dhadly MS, Temizer DH, Clifford JA, Yoshimuzi M, Quertermous T. Regulation of endothelin-1 gene expression by fos and jun. J Biol Chem 1991;266:19034-9.
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90. Textor SC, Canzanello VJ, Taler SJ, Romero JC, Porayko M, Wiesner RH, Krom R. Activation of the renin-angiotensin system and sustained endothelin release two years after liver transplantation [Abstract]. Circulation 1994;90:I-73. 91. Suzuki H, Sata S, Suzuki ¥, Takekashi K, Ishihara N, Shimoda S. Increased endothelin concentration in CSF from patients with subarachnoid hemmorrhage. Acta Neurol Scand 1990;81:553-4. 92. Kon V, Yoshioka T, Fogo A, Ichikawa I. Glomernlar actions of endothelin in vivo. J Clin Invest 1989;63:1762:6. 93. Shibouta Y, Suzuki N, Shino A, Matsumoto H, Terashita Z-I, Kondo K, Nishikawa K. Pathophysiological role of endothelin in acute renal failure. Life Sci 1990;46:1611-8. 94, Yamada K, Yoshida S. Role of endogenous endothelin on renal function in rats. Am J Physiol 1991;260:F34-8. 95. Kon V, Sugiura M, Inagami T, Harvie BR, Ichikawa I, Hoover RL. Role of endothelin in cyclosporine-induced glomerular dysfunction. Kidney Int 1990;37:1487-91. 96. Kircher KJ, Pollock DM, Opgenorth TJ, Kim CH, Sandberg SM, Edwards BS. Endothelin receptor antagonism does not reverse chronic cyclosporine induced renal vasoconstriction [Abstract]. Circulation 1994;90:L233.
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of the AMERICAN HEART JOURNAL for the preceding 5 years are maintained and are available for purchase from the publisher, Mosby, at a cost of $10.00 per issue. The following quantity discounts are available: 25% off on quantities of 12 to 23, and one-third off on quantities of 24 or more. Please write to Mosby-Year Book, Inc., Subscription Services, 11830 Westline Industrial Drive, St. Louis, MO 63146-3318, or call (800)453-4351 or (314)453-4351 for information on availability of particular issues. If unavailable from the publisher, photocopies of complete issues are available from University Microfilms International, 300 N. Zeeb Rd., Ann Arbor, MI 48106, (313)761-4700.
Endothelin is a naturally occurring polypeptide substance with potent vasoconstrictive actions. It was originally described as endotensin or endothelialcontracting factor in 1985 by Hickey et al., 1 who reported on the finding of a potent stable vasoconstricting substance produced by cultured endothelial cells. Subsequently, Yanagisawa et al.2 isolated and purified the substance from the supernatant of cultured porcine aortic and endothelial cells and then went on to prepare its complementary deoxyribonucleic acid (cDNA). This substance was renamed endothelin. Endothelin is the most potent vasoconstrictor known to date. Its chemical structure is closely related to certain neurotoxins (sarafotoxins) produced by scorpions and the burrowing asp (Atractaspis engaddensis). 3 Endothelins have now been isolated in various cell lines from several organisms. They are now considered to be autocoids or cytokines 4 because of their wide distribution, their expression during ontogeny and adult life, their primary role as intracellular factors, and the complexity of their biologic effects. In this review we discuss the biologic effects of endothelin and their receptors, the possible relation of endothelin to various pathophysiologic states, the role of endothelin as a disease marker, and the possible use of endothelin receptor blockade to treat various systemic illnesses. STRUCTURE
The superfamily of endothelins and sarafotoxins have two main branches with four members each. Endothelin is a polypeptide consisting of 21 amino
From the Department of Medicine, Division of Cardiology, The Albert Einstein College of Medicine-Montefiore Medical Center. Received for publication Nov. 30, 1994; accepted Jan. 10, 1995. Reprint requests: William H. Frishman, MD, Einstein College Hospital/ Montefiore Medical Center, 1825 Eastchester Rd., Bronx, NY 10461. AM HEARTJ 1995;130:601-10 Copyright © 1995 by Mosby-Year Book, Inc. 0002-8703/95/$3.00 + 0 4/1165063
acids. There are three closely related isoforms endothelin-1, endothelin-2, and endothelin-3 (ET1, ET2, and ET3, respectively), which differ in a few of the amino acid constituents (Fig. 1). The fourth member, called ET4 or vasoactive intestinal constrictor, is considered to be the murine form ofET2. 5 The endothelin molecules have several conserved amino acids, including the last six carboxyl (C)-terminal amino acids and four cysteine residues, which form two intrachain disulfide bonds between residues 1 and 15 and 3 and 11. These residues may have biologic implications particularly in relation to threedimensional structure and function. The main differences in the endothelin isopeptides reside in their amino (N)-terminal segments. There is a very high degree of sequence similarity between the two branches (approximately 60%) and within the constituent members of a branch (71% to 95%). PRODUCTION
Endothelin has been demonstrated to be produced from endothelial and nonendothelial cells. Factors known to release endothelin are shown in Table I. The synthesis of endothelins parallels that of the various peptide hormones in that a precursor polypeptide is sequentially cleaved to generate the active form (Fig. 2). Recently, endothelin-converting enzyme (ECE) was cloned. 5a ECE acts at an essential step in the production of active forms of endothelins. The fully formed molecule is then broken down into inactive peptides by as yet uncharacterized proteases. Some candidates are the lysosomal protective protein (deamidase) and enkephalinase (neutral endopeptidase EC 24.11). 6-s The regulation ofendothelin production occurs predominantly at the levels of transcription and translation. No storage vesicles containing endothelin have been identified. 9 The genes for the various endothelin isoforms have been sequenced and are found to be scattered in different chromosomes.Z° Current evidence suggests that they arose from a common ancestor by exon duplication 601
602
Tamir~a, F/~h/?~n, and Kb~Tr/~r Table
September 1995 American HeartJournal
I.
Factors known to release endothelin
Thrombin Transforming growth factor-~ Arginine vasopressin Hypoxia Phorbol ester Glucose Angiotensin II Cyclosporin Insulinlike growth factor Bombesin Cortisol Low-density lipeprotein cholesterol Hypercholesterolemia Changes in shear stress on vascular wall
Table ]]. R e c e p t o r affinities
Receptor ETA ETB ETC
~CO0S6b CO0-
Fig. 1. Amino acid sequences ofET1, ET2, vasoactive intestinal constrictor (VIC) (possibly mouse or rat analogue of ET2), and ET3 as well as sarafotoxin S6b, one of the peptide constituents of venom of burrowing asp Atractaspis engadclensis, termed sarafotoxins, which, with other members of this peptide family, have remarkable homology to endothelins. (From Kramer et al. Circulation 1992; 85:351.) followed by gene duplication with subsequent dispersion of the different genes. On thechromosome upstream to the genes, regions such as the AP1 motif, 11 nuclear factor-binding element, 12 and acutephase reactant regulatory elements have been identified and contain the motifs that interact with transcription factors and serve as potential points for gene control and modulation. ENDOTHELIN RECEPTORS
The receptors for endothelin have been isolated and their genes cloned. 13-15The receptors are classified on the basis of their affinity for the various endothelin isoforms. So far two receptors, called ETA and ETB, have been well characterized. The gene for a third receptor could not be isolated by hybridiza-
Affinity ET1 > ET2 > ET3 ET1 = ET2 = ET3 ET3 > ET1
tion techniques with the cDNA of ETB, suggesting that it is less homologous to the other two receptors. In h u m a n beings ETA and ETB receptors exhibit significant sequence similarity, with about 63% amino acid identityJ 6 Evidence also points to the presence of subtypes among the various receptors.17, 18 The receptors appear to be glycoproteins; the sugar moiety is a possible important constituent for ligand interactions. 14 The ETA receptors probably recognize the tertiary structure of both the N-terminal and the C-terminal segments, whereas the ETB receptors recognize predominantly the Cterminal parts, explaining the differences in affinities of the two receptors for the isoforms (Table II). 9 The endothelin receptors belong to the rhodopsin superfamily, which also includes the B1, B2, 5HT1, 5HT2, V1, and V2 receptors. Members of this family are characterized by the presence of seven transmembrane segments in the receptor that span the membrane; their action is mediated by G proteins. There are many types of G proteins: inhibitory, stimulatory, and others. The binding of the ligand to the receptor induces an allosteric effect that causes corresponding changes in the interaction with the G proteins. It is thought that the third intracellular loop of the receptor is the site responsible for interacting with the G proteins. The exact residues of the receptor that interact with the ligand are not clearly known, and mutational studies are in progress to identify the responsible residues.
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Tam#'isa~ Frishman, and Kumar 603
Fig. 2. Pr~te~yticpr~cessing~fprepr~end~the~inandproendothelininbiosynthesisofmatureET1.From Highsmith HF et al. (Reproduced, with permission, from the Annual Review of Physiology, Volume 54, pages 257-77, © 1992 by Annual Reviews Inc.) The differential distribution of the endothelin receptors in various tissues is responsible for the multiplicity of actions attributed to endothelin (Table III). The binding of endothelin to its receptor is very tight, with relatively slow dissociation, which may result in prolonged action. An important factor to consider is that some of the effects seen in vitro may not be biologically relevant to events in vivo. 19 ETA receptors have been found predominantly in the heart, blood vessels of the brain and vascular smooth muscle, whereas the ETB receptors are more widely distributed and predominate in the kidney, uterus, central nervous system, and endothelial cells. MECHANISM OF ACTION
The binding of endothelin to its receptor has several effects (Fig. 3). Activation of phospholipase C causes hydrolysis of phosphatidylinositol, forming inositol-l,4,5-trisphosphate (IP3) and diacylglycerol. The newly formed IP3 causes release of calcium from the sarcoplasmic reticulum storage sites. It also facilitates the entry of extracellular calcium by dihydropyridine-sensitive voltage channels. The total intracellular free calcium concentration increases; the initial increase reflects the release from cellular stores, and the latter increase reflects net inward calcium entry across the cell membrane. 2°-23 The greater concentration of intracellular calcium often associates with calmodulin and modulates diverse processes such as neurotransmitter release, secretion, and activation of enzymes including myosin light-chain kinases that activate cellular contractile machinery and other mechanisms. 24 Nifedipine and
Fig. 3. Intracellular signal transduction pathways activated by endothelins (ETs). Activated ET receptor stimulates phospholipase C (PLC) and phospholipase A2 (PLA2). Activated ET receptor also stimulates voltage-dependent calcium channels (VDC) and probably receptor-operated calcium channel (ROC). Inositol triphosphate (IP3) elicits release of calcium ion from caffeine-sensitive calcium store. Protein kinase C (PKC) activated by diacylglycerol (DG) sensitizes contractile apparatus. Increased concentration of intracellular free calcium ion ([Ca2+]~induces contraction. Cyclooxygenase products (prostacyclin [PGI2], prostaglandin E2 [PGE2], and thromboxane A2 [TXA2]) modify contraction. G, G protein; IP2, inositol biphosphate; IP3, inositol triphosphate; PIP2, phosphatidyl inositol biphosphate. (From Masaki T et al. Circulation 1991;84: 1460.)
other calcium-channel blocking agents can inhibit the influx of extracellular calcium, thereby antagonizing some of the actions of endothelin. 25-27 The sustained release of DAG causes prolonged
September 1995 American Heart Journal
604 Tamirisa, Frishman, and Kumar Table III. Two distinct pharmacologic activities of endothelins Receptor affinity rank order
System 1 (ETAtype): ET1 > ET3
Tissue or organ and species
Vascular smooth muscle cells Airway smooth muscle (human) Uterine smooth muscle (rat) Cardiac muscle (neonatal rat) Adrenocorticalcells (zona glomerulosa, bovine) C6 glioma cells (rat) Fibroblast cells (human, mouse) Osteoblast cells (rat)
System 2 (ETBtype) ET1 = ET3
Vascular smooth muscle cells (rat) Mesenteric artery (rat) Renal pelvis smooth muscle (human) Stomach (rat) Blood (rabbit) Cerebral cortex,cerebellum(rat) Adrenocorticalcells (bovine) Astrocyte (rat) Mesangium cells (rat)
Assay
Pressor effectin vivo, contraction,PI generation, binding assay Contraction Contraction, PI generation, binding assay PI generation, binding assay Stimulation of aldosterone secretion, binding assay PI generation, binding assay Calcium transient,* binding assay DNA synthesis, PI generation, binding assay Initial depressor response Transient relaxation, NO release Contraction Ulcerogenicity Inhibition of platelet aggregation ex vivo PI generation Binding assay PI generation, calcium transient PI generation,binding assay
Endothelin-induced responses may be divided into two groups according to pharmacologic Potency Rank order among the three endothelin isopeptides.
DNA, Deoxyribonucleic acid; NO, nitric oxide (enothelium-derived relaxing factor) PI, phosphoinositide (inositol phosphate). *Transient increase in intracellular calcium evoked by endothelins. (Adapted from Sakurai T, Goto IL Drugs 1993;46:795-804.)
activation of protein kinase C (PKC), 28 which, by causing phosphorylation of various proteins, m ay result in activation or inactivation of proteins and the regulation of gene expression. Endothelin has also been noted to cause increase in intracellular pH, perhaps by stimulating the sarcolemmal sodium ion-hydrogen ion antiporter. This increased intracellular pH can enhance the sensitivity of the myofilaments to calcium, thereby augmenting contraction even without changing the calcium concentration.29, 30 It has also been noted t h a t the increase in intracellular pH can by itself stimulate hypertrophic and mitogenic responses in different cells. 31, 32 The binding of endothelin to its receptor also m ay alter ion-channel permeability (such as t ha t of the voltage-dependent L-type calcium channels by G proteins) and cause activation of other secondary messengers (such as production of prostanoids by activating phospholipase A)33 and release of nitric oxide. 34-36Nitric oxide has been shown to antagonize some of the effects ofendothelin on vascular smoothmuscle contraction, perhaps by decreasing endothelin release from endothelial cells. 37 In addition, experiments have revealed a competitive inhibition of ET1 binding to its receptors in cardiac membranes of rats by potassium-channel opening agents. These agents have been shown to exert cardioprotective
action after experimental myocardial infarction in rats by suppressing cardiac arrhythmias, which may result in part from inhibition of the effects of cardiac endothelin. It has been suggested t hat ET1 m a y close adenosine triphosphate-sensitive potassium channels.38-40 PATHOPHYSIOLOGIC ROLES OF ENDOTHELIN Systemic hypertension. Endothelin is the most potent vasoconstrictor known to date and has an exceptionally long duration of physiologic action. 9 The influence of endothelin in maintaining normal blood pressure and its role in the cause of systemic hypertension remain unclear. 41 Intravenous injections of endothelin in animals cause a transient decrease in systolic blood pressure (ETB) followed by a prolonged pressor response (ETA). 2, 11 The vasoconstrictor action is mediated by ETA receptors in the vascular smooth muscle, whereas the predominant vasodilation effect is mediated by the ETB receptors on the endothelial cells t h a t cause release ofprostacyclin and nitric oxide. Therefore the overall predominant hemodynamic effect of endothelin in a given organ depends on the receptor type being stimulated, its location, and its relative abundance. Angiotensin II has been found to increase endothelin concentrations (Table IV) in vitro from endo-
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thelial cells, suggesting one mechanism by which angiotensin-converting-enzyme (ACE) inhibition could function in vivo. 42 ACE inhibitors also can indirectly interfere with endothelin: increased concentrations of bradykinin decrease endothelin release (by acting through bradykinin 2 receptors, stimulation of which cause increased nitric oxide release). ACE inhibitors can cause regression of intimal hyperplasia, whereas other antihypertensive drugs are ineffective in this regard. 43 The significance of plasma endothelin concentrations in hypertension has been subject to controversy. Endothelin has been implicated as the cause for cyclosporine-induced hypertension. After the development of new specific and sensitive assays for endothelin, it was shown that there were no significant differences in the endothelin concentrations of patients with normal blood pressure and with hypertension. 44 The previously reported higher plasma concentrations ofimmtmoreactive endothelin in subjects with hypertension is now attributed to cross reactivity of the older assays. On the other hand, an increased sensitivity to available endothelin has been reported in some studies. 45, 46 The plasma concentrations of endothelin tend to increase with age and are higher in men than women in both normotensive and hypertensive subjects. 44 Atherosclerosis. Hypercholesterolemia leads to profound alterations in the regulation of vascular tone. Low-density lipoprotein accumulates in vascular walls in hypercholesterolemia. Experimental studies have shown that oxidatively modified low-density lipoproteins induce the production of ET1 by h u m a n macrophages and can increase endothelin formation and release from endothelial cells. 47, 4s High plasma concentrations of endothelin have been described in patients with atherosclerosis. 49 One of the early mechanisms of atherosclerosis includes the migration of monocytes into the intimal layer of blood vessels, where they become activated and stimulate the release of multiple factors. It has been demonstrated in vitro that endothelin can serve as a chemotactic agent for monocytes, and therefore its role as an initiator of the atherosclerotic process has been strengthened. Endothelin also may stimulate the migration and proliferation of vascular smooth-muscle cells, t h e r e b y sustaining the atherosclerotic p r o c e s s . 27, 50, 51 Calcium-channel blocking agents have been shown to reduce the chemotatic movements of monocytes, perhaps by interfering with calcium ion fluxes. Myocardial ischemia. Myocardial ischemia can enhance the release of endothelin by cardiomyocytes and increase its vasoactive effects. Infusion of the
Tamirisa, Frishman, and Kumar 605 Table iV. Conditions associated with increased circulating
endothelin in blood Acute myocardial infarction Allograi~ rejection Atherosclerosis Cold exposure Coronary spasm Cardiomyopathy Disseminated intravascular coagulation Diabetes mellitus Exercise Essential hypertension Hemodialysis Hypertension of pregnancy Ischemic heart disease Preeclampsia Pregnancy Pulmonary hypertension Postural changes Raynaud's phenomenon Stable chronic renal failure Transplantation of organs Trauma Uremia Subarachnoid hemorrhage Surgery Sepsis Scleroderma Shock
ET1 isoform directly into the coronary circulation of animals results in the development of myocardial infarction, with impaired ventricular functioning and the development of arrhythmias. 52 Endothelin has been shown to lower the threshold for ventricular fibrillation in dogs. 53 An increase in ET1 has been observed in cardiac tissue after experimental myocardial infarction in rats, and pretreatment with an antiendothelin ~-globulin in this model can reduce infarct size by as much a s 40%. 54 Infusion of ETAreceptor antagonist drugs before an ischemic insult can also reduce infarct size in animals. 55 Plasma endothelin concentrations and urinary endothelin excretion are increased in patients with myocardial infarction 56 and in vasospastic angina but not in stable or unstable angina. 51, 54, 57 Patients who undergo successful thrombolysis with early reperfusion have lower endothelin concentrations than subjects who do not have early reperfusion. Plasma endothelin concentrations can also predict hemodynamic complications in patients with myocardial infarction. 5s Patients with the highest plasma endothelin concentrations after myocardial infarction have the highest creatine phosphokinase (CPK) and CPK MB-isoenzyme concentrations and the lowest angiographically determined ejection fractions. 59 It has also been suggested that the serum
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concentration of endothelin can be used as a prognostic indicator of survival after acute myocardial infarction. Left ventricular function and congestive heart failure.
Endothelin exhibits potent inotropic activity in isolated hearts, cardiac muscle strips, isolated cells, and instrumented intact animals. 6° High-affinity receptors for endothelin have been demonstrated in the atria and the ventricles. 61-63 Intravenous administration of the ET1 isoform produces delayed prolonged augmentation of left ventricular performance in addition to its biphasic vasoactive effects of transient vasodilation followed by sustained vasocontraction. 60 Endothelin is a potent secretogne of atrial natriuretic factor, which is a naturally occurring antagonist of endothelin. 64 The ETA receptor appears to mediate endothelin's actions of vasoconstriction and the stimulation of atrial natriuretic factor secretion, and the ETB receptor mediates endothelin-induced vasodilation and activation of the renin-angiotensin-aldosterone system. Urinary water excretion is mediated through both receptors, but sodium excretion is mediated through the ETA receptor. Increased concentrations of endothelin described in patients with congestive heart failure 65, 66 are predictive of increased mortality r i s k . 66 It also has been suggested that increased concentrations of endothelin may play an important role in the increased systemic vascular resistance observed in congestive heart failure. 65 Increased endothelin concentrations have also been observed in the plasma and hearts of cardiomyopathic Syrian hamsters 67 and in the cells of endothelial cells infected with Trypanosoma cruzi in experimental Chagas' cardiomyopathy. 6s There is early clinical evidence that treatment with ETA receptor antagonists and ECE inhibitors can influence favorably the course of h u m a n heart failure. 69 ACE inhibitors may also benefit patients with heart failure because of their antiendothelin actions. 7° Pulmonary hypertension. Expression of ET1 in the lung has been studied 7°a by immunocytochemistry and hybridization in situ in specimens from patients with pulmonary hypertension of primary or secondary causes. In contrast to normal lung, specimens from patients with pulmonary hypertension exhibit abundant ET2 immunostaining, particularly over endothelium of markedly hypertrophied muscular pulmonary arteries and plexogenic lesions. Endothelin has been suggested as a potent vasoconstrictor and growth-promoting factor in the pathophysi-
September 1995 American Heart Journal
ologic mechanisms of pulmonary hypertension. 71-74 In animals, an orally active ETA and ETB receptor blocking agent, bosentan, has been shown to prevent hypoxia-induced pulmonary hypertension and pulmonary artery remodeling. 72, 75 Endothelin has been proposed to be a local modulator for oxygen to cause closure of the ductus arteriosus after birth. 76 Increased endothelin concentrations are found in various congenital heart disorders associated with left-to-right shunts. 77 Ventricular and vascular hypertrophy. Endothelin increases DNA synthesis in vascular smooth-muscle ceils,7S, 79 cardiomyocytes,80 fibroblasts, glial cells, mesangial cells, and other cells; causes expression of protooncogenes; causes cell proliferation27; and causes hypertrophy, sl It acts in synergy with various factors such as transforming growth factor, epiderreal growth factor, platelet-derived growth factor, basic fibroblast growth factor and insulin to potentiate cellular transformation and replication. This synergy suggests that all of these factors act through common pathways involving PKC and cyclic adenosine monophosphate. Endothelin per se may not be a direct mediator of angiogenesis but may function as a comitogenic factor. 27 The role of endothelin as a mitogen for vascular smooth-muscle cells is unclear. It has been found that externally added endothelin to the cultured rat aortic smooth-muscle cell does not induce proliferation, but when the same cells are transfected with endothelin producing plasmids, cell lines that produce the greatest amount of endothelin show the greatest proliferation, suggesting an autocrine action. In addition, this effect is significantly reduced by ETA-specific antagonists. 82, s3 Increased protein synthesis induced by endothelin may play a role in myocardial hypertrophy, s4,s5 Marked increase in prepro-ET1 messenger ribonucleic acid and mature ET1 has been found in the ventricles of rats in which concentric hypertrophy was induced experimentally, s6, s7 Regulation ofgene expression as shown by increased levels of c-fos, c-myc, fra 1, jun b, and other genes has been found in cells stimulated by endothelin, s°, s5, 88 The regnlation of ET1 gene transcription by fos andjun oncogenes, 89 which in turn are regulated by ET1, creates a loop, which makes matters more complex, Neointima formation after vascular wall trauma. The efficacy of coronary angioplasty is limited by the high incidence ofrestenosis. ET1 induces cultured vascular smooth-muscle cell proliferation by activation of the ETA-receptor subtype, a response that normally
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is attenuated by an intact, functional endothelium. In addition, ET1 also induces the expression and release of several protooncogenes and growth factors that modulate smooth-muscle cell migration, proliferation, and matrix formulation. In addition to inhibiting smooth-muscle cell proliferation in vitro, endothelin-receptor antagonism with SB 209670 ameliorates the degree of neointima formation observed after rat carotid artery angioplasty. The observations raise the possibility that ET1 antagonists will serve as novel therapeutic agents in the control of restenosis. 83 AIIograft rejection. ET1 concentrations are increased in patients with graft rejection and arteriosclerosis. This increase in endothelin activity may contribute to altered microvascular hemodynamics and remodeling and to the long-term hypertension and nephrotoxicity seen in patients undergoing transplantation. 90 Cerebrovascular disease. Laboratory and clinical investigations suggest that endothelin play a role in the pathogenesis of delayed cerebral vasospasm in subarachnoid hemorrhage and ischemic stroke. High concentrations of endothelin have been demonstrated in the cerebral spinal fluid of these patients. 91 Endothelin-receptor inhibitors have been suggested as treatments for patients with subarachnoid hemorrhage to influence cerebral vasoconstriction and delaying neuronal death. Renal function, acute renal failure, and nephrotoxicity. Endothelin can cause increased renal vascular resistance, decreased renal blood flow, and decreased glomerular filtration, effects that resemble those seen in renal failure. In some studies, the infusion of antiendothelin antibodies has been found to exert a renal protective effect in ischemia 9294 and in cyclosporine nephrotoxicity. 95 However, in a recent study, the use of a specific ETA receptor blocking agent was found not to reverse chronic cyclosporine-induced vasoconstriction. 96 ENDOTHELIN ANTAGONISTS
Active research and drug development programs are aimed at investigating endothelin inhibitors. Preventing the possible pathophysiologic effects of endothelin at their receptors with selective antagonists is a new frontier in pharmacotherapy. Several antagonists to endothelin have been discovered in recent years (Table V). The original receptor antagonists were isolated from the cultured broth of Streptomyces misakiensis. Some of the agents are receptor specific for the ETA or the ETB subtype, but
Tamirisa, Frishman, and Kumar 607 Table V. Endothelin receptor antagonists ETA
ETB
Nonspecific
BQ 123 BQ 153 FR 139317 Asterric acid BMS 182874
IRL 1038 BQ 788 RES 701-1
PD 145065 PD 142893 Cchinmicins I, II, and III Ro 462005 Bosentan SB 209670 Phosphoramidon (ECE inhibitor)
Table VI. Nonspecific endothelin antagonists ECE inhibitors Angiotensin-converting-enzyme inhibitors Angiotensin II receptor blocking agents Calcium-entry blocking agents Potassium-channel opening agents Adenosine Nitroglycerin These antagonists interfere with release or modify action of endothelin.
others are non-specific. There also are other classes of drugs (Table VI) that interfere with endothelin release or modify its actions. Efforts are now underway to customize specific antagonists, and according to the known structure of endothelin, to modify key amino acids to create innovative compounds. Another innovative approach is to control endothelin action by interfering with the ECE essential for the production of the active compounds. The use of endothelin antagonists that can be used intravenously or orally will help to elucidate the mechanisms by which endothelin mediates its effects. These drugs m a y also be of use in the treatment of patients with systemic hypertension, myocardial ischemia, restenosis after angioplasty, congestive heart failure, subarachnoid hemorrhage, renal failure (radiocontrast induced), allograft rejection, or sepsis. Clinical trials of the use of endothelin antagonists for many of these conditions are now in progress. SUMMARY
Endothelin is the most potent mammalian vasoconstrictor yet discovered. Its three isoforms play leading roles in regulating vascular tone and causing mitogenesis. The isoforms bind to two major receptor subtypes (ETA and ETB), which mediate a wide variety of physiologic actions in several organ systems. Endothelin may also be a disease marker or an etiologic factor in ischemic heart disease, atheroscler0-
,
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sis, congestive heart failure, renal failure, myocardial and vascular wall hypertrophy, systemic hypertension, pulmonary hypertension, and subarachnoid hemorrhage. Specific and nonspecific receptor antagonists and ECE inhibitors that have been developed interfere with endothelin's function. Many available cardiovascular therapeutic agents, such as angiotensin-converting-enzyme inl~ibitors, calciumentry blocking drugs, and nitroglycerin, also may interfere with endothelin release or may modify its activity. The endothelin antagonists have great potential as agents for use in the treatment of a wide spectrum of disease entities and as biologic probes for understanding the actions of endothelin in human beings. REFERENCES
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40. Kerr MJ, Wilson R, Shanks RG. Suppression of ventricular arrhythmias after coronary artery ligation by pinacidil, a vasodilator drug. J Cardiovasc Pharmacol 1985;7:875-83. 41. Donckier J, Stoleru L, Hayashida W, Van Mechelen H, Galanti L, Ketelslegers J-M, Pouleur H. Role of endogenous endothelin-1 in experimental hypertension [Abstract]. Circulation 1994;90:I-387. 42. Ciafre SA, D'Armiento FP, DiGregorio F, Colasanti P, DiBenedette A, Langella A, Di Ieso N, Liguori A, Colasanti R, Napoli C. Angiotensin II stimulates endothelin-1 release from h u m a n endothelial cells. Recenti Prog Med 1993;84:248-53. 43. Powell JS, Clozel JP, Muller RK, Kuhn H, Hefti F, Hosang M. Inhibitors of angiotensin converting enzyme prevent myointimal proliferation after vascular injury. Science 1989;245:186-8. 44. Miyauchi T, Yanagisawa M, Iida K, Ajisaka R, Suzuki N, Fujino M, Goto K, Masaki T, Sugishita Y. Age- and sex-related variation of plasma endothelin-1 concentration in normal and hypertensive subjects. AM HEARTJ 1992;123:1092-3. 45. Catelli DeCarvalho MH, Nigro D, Scivolette R, Barbeiro HV, Aparecida deOliveira M, deNucci G, Fortes ZB. Comparison of the effect of endothelin on microvessels and macrovessels in Goldblatt II and deoxycorticosterone acetate-salt hypertensive rats. Hypertension 1990;15(suppl I):I68-71. 46. Tomobe Y, Miyauchi T, Saito A, Yanagisawa M, Kimura S, Goto K, Masaki T. Effects ofendothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats. Eur J Pharmaco11988;152:373-4. 47. Boulanger CM, Tanner FC, Bea ML, Hahn AWA, Werner A, Luscher TF. Oxidized low density lipoprotein induces mRNA expression and release of ET from h u m a n and porcine endothelium. Circ Res 1992; 70:1191-7. 48. Martin-Nizard F, Houssaini HS, Lestavel-Delattre S, Duriez P, Fruehart JC. Modified low density lipoproteins activates macrophages to secrete ir-ET. FEBS Lett 1991;293:127-30. 49. Lerman A, Edwards BS, Hallett JW, Heublein DM, Sandberg SM, Burnett JC Jr. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis: N Engl J Med 1991;325:997-1001. 50. Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular diseases. Circ Res 1986;58:427-44. 51. Haynes WG, Webb DJ. The endothelin family: local hormones with diverse roles in health and disease? Clin Sci 1993;84:485-500. 52. Karwatowska-prokopezuk E, Wennmalm A. Effects of endothelin on coronary flow, mechanical performance, oxygen uptake and formation of purines and on outflow of prostacyclin in the isolated rabbit heart. Circ Res 1990;66:46-54. 53, Salvati P, Chierchia S, Dho L, Ferrario RG, Parenti P, Vicedomini G, Patrone C. Proarrhythmic activity ofintracoronary endothelir/in dogs: relation to the site of administration and to changes in regional flow. J Cardiovase Pharmacol 1991;17:1007-14. 54. Watanabe T, Suzuki N, Shimamoto N, Fujino M, Imada A. Contribution of endogenous endothelins to the extension of myocardial infarct size in rats. Circ Res 1991;69:370-7. 55. Grover GJ, Dzwonczyk S, Parham CS. The endothelin-1 receptor antagonist BQ-123 reduces infarct size in a canine model of coronary occlusion and reperfusion. Cardiovasc Res 2993;27:1613-8. 56. Omland T, Lie RT, Aakvaag A, Aarsland T, Dickstein K. Plasma endothelin determination as a prognostic indicator of i year mortality after acute myocardial infarction. Circulation 1994;89:1573-9. 57. Ray SG, McMurray JJ, Morton JJ, Dargie HJ. Circulating endothelin in acute ischemic syndromes. Br Heart J 1992;67:383-6. 58. Leehleitner P, Genser N, Mair J, Maier J, Artner-Dworzak E, Dienstl F, Puschendorf B. Endothelin-1 in patients with complicated and uncomplicated myocardial infarction. Clin Investig 1992;70:1070-2. 59. Lechleitner P, Genser N, Mair J, Maier J, Artner-Dworzak E, Dienstl F, PuschendorfB. Plasma immunoreactive endothelin in the acute and subacute phases of myocardial infarction in patients undergoing fibrinolysis. Clin Chem 1993;39:955-9. 60. Ohno M, Li W, Cheng C-P. Effects of endothelin-1 on lei~ ventricular performance in conscious dogs: assessment by pressure-volume analysis [Abstract]. Circulation 1994;90:I~16. 61. Gu X-H, Casley D, Nayler W. Specific high affinity binding sites for 125I-labelled porcine endothelin in rat cardiac membranes. Eur J Pharmacol 1989;167:281-90. 62. Galron R, Kloog Y, Bdolah A, Sokolovsky M. Functional endothelin/sa-
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September 1995 American Heart Journal
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