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Endothelins
Chapter 190
Endothelins Takashi Miyauchi and Katsutoshi Goto
ABSTRACT A potent vasoconstrictor peptide, endothelin (endothelin-1), was discovered from culture supernatant of porcine endothelial cells in 1988. From human genomic DNA analysis, two other family peptides, endothelin-2 and endothelin-3, were found. They showed different effects and distribution, suggesting that each peptide may play separate roles in different organs. Endothelins act via the activation of two receptor subtypes, ETA and ETB receptors, both of which are coupled to various GTPbinding proteins depending on cell types. Endogenous ET-1 may be involved in the progression of various cardiovascular diseases, and ET antagonists are currently used clinically in the treatment of patients with pulmonary hypertension and are considered to have further target diseases such as systemic hypertension, heart failure, cardiac diseases, renal diseases.
DISCOVERY Vasoconstriction dependent on or enhanced by intact endothelium has been observed in response to various chemical and physical stimuli such as norepinephrine, thrombin, hypoxia, increased transmural pressure, mechanical stretch. These observations lead to the speculation that endothelial cells may release certain vascular constricting substance(s), endothelium-derived contracting factors (EDCF). In 1985, Hickey et al. attempted to test the biological activity of the culture medium of bovine aortic endothelial cells on isolated porcine coronary arteries, and they found that the culture supernatant contained peptide-like factor(s), which triggered a slowly developing and long-lasting contraction of the coronary arteries. Based on gel chromatography analysis, they suggested that this particular EDCF is a peptide with a molecular mass of approximately 8500 Da.8 At about the same time, Gillespie et al. also detected vascular constricting activity in the culture supernatant of porcine aortic endothelial cells. They subsequently reported that the activity was increased when the cultured endothelial cells were stimulated by thrombin, and they suggested that this EDCF is a peptide with a molecular mass of approximately 3000 Da.19 1402
In 1987, we performed experiments similar to those of Hickey et al. and confirmed that the supernatant from confluent monolayer cultures of porcine aortic endothelial cells contained a slowly developing and long-lasting vascular constricting factor(s), peptidic in nature, because the vascular constricting activity was abolished by pretreatment of the conditioned medium with trypsin. The activity was also detected in serum-free conditioned medium, and no appreciable change in activity was observed even after long-term (2–3 weeks) maintenance of the endothelial cell culture in serum-free condition. The successful attempt at serum-free maintenance and detection of vascular constricting activity prompted us to isolate and purify the active peptide in the supernatant because of the absence of interference with proteins and/or peptides in the serum itself. Cells isolated from porcine thoracic aortas and grown to a confluent monolayer were maintained in serum-free minimum essential medium. The medium was changed every 5 days, and the conditioned medium was pooled at −20 °C. The pooled conditioned medium was first centrifuged at 1000 g for 20 min, and subsequently, the supernatant was desalted and concentrated. The concentrated medium was loaded onto an anion-exchange column and eluted by applying a linear gradient of NaCl. The vascular constricting activity of the eluent was assayed by adding a small amount of each fraction directly into a muscle chamber where a helical strip of porcine coronary artery with the intima denuded was suspended, and the active fraction was collected. The active fraction was subjected to reversedphase high-performance liquid chromatography (HPLC) and elution with a linear gradient of acetonitrile, and the vascular constricting activity of each fraction was similarly assayed. A second trial of reversed-phase HPLC enabled us to purify the active component. Approximately 3 nmol of the final product was obtained, which was just enough to perform subsequent amino acid analysis. The purified peptide was subjected to amino acid sequence analysis by means of an automated gas-phase peptide sequencer and carboxy-terminal analysis by hydrazinolysis (Edman’s reaction). As a result, the peptide was revealed to be composed of 21 amino acid residues with Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00190-1 Copyright © 2013 Elsevier Inc. All rights reserved.
SECTION | XIV Handbook of Biologically Active Peptides: Cardiovascular Peptides
free amino- and carboxy-termini. At first, the carboxyterminal amino acid (tryptophan) was not detected, and a 20 amino acid peptide synthesized artificially did not exhibit any biological activity. It was soon recognized that the structure of tryptophan (indole ring) is easily degraded during Edman’s reaction. The peptide comprising 21 amino acid residues subsequently synthesized did exhibit vascular constricting activity identical to that of natural peptide. The four cysteine residues at the amino-terminal portion were found to form two intramolecular disulfide bonds, the topological analysis of which revealed that the arrangement of disulfide bonds is a coaxial form (1–15 and 3–11). Because it was originally discovered from the culture supernatant of endothelial cells, the peptide was termed “endothelin.”27
STRUCTURE OF THE mRNA/GENE Based on the amino acid residues 7–20 of endothelin, several kinds of oligonucleotides were synthesized according to the mammalian codon usage statistics. Using one of the synthesized oligonucleotides as a single “optimal” DNA probe, approximately 2 × 106 clones were screened from aλgt10 cDNA library constructed for porcine endothelial cell mRNA, and 38 hybridization-positive clones were soon identified. Four of these clones were subjected to further characterization, and finally, a complete nucleotide sequence of porcine preproendothelin cDNA and the deduced amino acid sequence were determined.
PROCESSING OF THE PRECURSOR The 203 residue porcine preproendothelin contains 19 residues of deduced N-terminal amino acid sequence, being characteristic of a secretory signal sequence, that is, a hydrophobic core followed by residues with small polar side chains. As anticipated, paired basic amino acid residues Lys51-Arg52, which are recognized by the usual processing endopeptidase, furin (see the furin chapter in Peptide Biosynthesis/Processing section of the book), directly precede the endothelin sequence, but no dibasic pair is found thereafter until Ara92-Arg93. This indicates that the intermediate peptide of 39 amino acids, called big endothelin (Cys53Arg92), may be first generated, and subsequently, mature endothelin may be generated via the unusual proteolytic processing between Try73 and Val74 in big endothelin by an endopeptidase exhibiting chymotrypsin-like specificity, which was putatively termed “endothelin-converting enzyme (ECE).” This presumptive pathway of endothelin biosynthesis in endothelial cells was confirmed later. The human preproendothelin cDNA was soon cloned, and its sequence of 212 amino acid residues containing big endothelin of 38 amino acid sequences was determined (Fig. 1). The cDNA of ECE was also cloned, and it was revealed that ECE is a 785 amino acid metalloprotease containing a
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single membrane-spanning sequence with only a 56-residue N-terminal cytoplasmic tail and an extracellular C-terminal of 681 amino acid residues that contains the catalytic domain.26 Amino acid residues 593–601 match the highly conserved consensus sequence of a zinc-binding motif, HEXXH, which is shared by many known metalloproteases. The ECE protein has ten predicted sites for N-glycosylation in the extracellular domain, suggesting that ECE behaves as a highly glycosylated protein.
DISTRIBUTION OF THE mRNA/PEPTIDE IN THE CARDIOVASCULAR SYSTEM Southern blot analysis of human genomic DNA under low hybridization stringency with a 42-mer synthetic oligonucleotide probe corresponding to amino acid residues 7–20 of endothelin, showed that three different restriction fragments were always detected regardless of the restriction endonucleases used. The nucleotide sequences encoding amino acid residues of the three endothelins are highly conserved among the three genes, with 77–82% of the nucleotide residues being identical. By contrast, the nucleotide sequences upstream from the mature peptides are very poorly conserved. These observations suggest that although the three genes are evolutionally relatively distant from each other, the genes evolved from a common ancestral gene under strong pressure to preserve mature endothelin sequences. The three peptides were designated endothelin-1, endothelin-2, and endothelin-3.10 Endothelin-1 is the original peptide corresponding to that detected in the culture medium of porcine aortic endothelial cells. Although vascular endothelial cells are the major source of endothelin-1, Northern blot analysis revealed that the genes encoding the three endothelin isopeptides are expressed with different patterns in a wide variety of cell types including cardiac myocytes, vascular smooth muscle cells, pituitary cells, macrophages, and mast cells, suggesting that the peptides may participate independently in complex regulatory mechanisms in various organs. The conservation of numerous endothelin-related genes was also observed in several mammalian species examined, including humans, pigs, and rats. Further, four cardiotoxic peptides highly homologous to endothelins, sarafotoxins (S6a–S6d), were isolated from the snake venom of Atractaspis engaddensis. Endothelin-like immunoreactivity was also found in several species of invertebrates and fishes, indicating that endothelins found in humans seem to have a long evolutionary history. Since endothelin-1 was discovered first and showed a wide variety of actions not only on the cardiovascular system but also on various other tissues, much information has accumulated on endothelin-1 compared with the two other peptides. The method for measurement of endothelin-1 levels in plasma or various tissues by means of
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Chapter | 190 Endothelins
FIGURE 1 The biosynthesis, intracellular processing of endothelin-1 (ET-1) from preproET-1. Endothelin-converting enzyme (ECE) specifically catalyzes the conversion of big ET-1 to ET-1. Cys–Cys represents the intramolecular disulfide bond.
radioimmunoassay (RIA) or enzyme immunoassay (EIA)25 was established soon after the discovery of endothelin-1. RIA uses only one kind of antibody against endothelin-1, whereas EIA uses two kinds of antibodies, each recognizing the N-terminal and C-terminal portions of endothelin-1, respectively. At an early stage, the reported plasma concentrations of endothelin-1 in humans were fairly divergent, ranging from 0.2 to 18.5pg/ml in healthy subjects. To avoid unnecessary confusion, the reason for the difference in the values of plasma endothelin-1 level was examined thoroughly and, as a result, it was generally agreed that the divergence originated mostly from differences in the specificity of the antibodies used and in the recovery rate during extraction of endothelin-1 from plasma or tissues. It is now accepted that the plasma concentration of endothelin-1 in healthy subjects is 1.0–2.0pg/ml,13–16 which is at least one order of magnitude lower than that of circulating human natriuretic peptides and several times less than that
of angiotensin II. Plasma concentrations of endothelin-1 increase drastically in the case of various cardiovascular diseases.13–16 Endothelin-1 may be released in both luminal and abluminal directions from endothelial cells in vivo. Luminal released endothelin-1 may be diluted by the blood stream, and its circulating concentration (1.0–2.0pg/ml) is below the threshold concentration producing vasoconstriction. Although the exact concentration in the abluminal space (vascular smooth muscle surface) is not known, endothelin-1 is more likely to be a locally acting rather than a circulating hormone. When endothelin-1 production and secretion are increased drastically in certain sites under pathophysiological conditions, severe local vasoconstriction might be anticipated. When endothelin-1 is intravenously administered as a bolus, it disappears quite rapidly from the blood stream with a half-life of a few minutes. This rapid removal of endothelin-1 from the circulation results from uptake into
SECTION | XIV Handbook of Biologically Active Peptides: Cardiovascular Peptides
various tissues, including the lung, kidney, spleen, and liver. The lung seems to be one of the most important tissues for uptake, because approximately 60% of endothelin-1 is removed after a single passage through the pulmonary circulation. In lung tissues, the ETB receptor is highly expressed and endothelin-1-ETB receptor complex may be taken up into cells through an internalization process and degraded by lysozomal enzymes, for example, aspartic proteases. Neutral endopeptidase and lysozomal cathepsin G are also concerned with enzymatic degradation of endothelin-1. Despite such a rapid disappearance from the circulation, the blood pressure–elevating effect of endothelin-1 continues for an extremely long period; stabilization of the endothelin-1-ETA receptor complex may contribute, but the exact reason for this phenomenon is unknown.
RECEPTORS AND THEIR DISTRIBUTION Endothelin-induced responses can be divided into two major groups, according to the pharmacological potency order of the three isopeptides. In the first group of responses, including vascular constriction, broncho-constriction, uterine smooth muscle constriction, and stimulation of aldosterone secretion, endothelin-1 and endothelin-2 act as far more potent agonists than endothelin-3 do. In the second group, which includes endothelium-dependent vascular relaxation, astrocyte proliferation, and inhibition of ex vivo platelet aggregation, the three isopeptides possess almost equal potency. These findings suggest that there are at least two distinct endothelin receptors mediating these two distinct groups of pharmacological responses. The existence of multiple receptors was also supported by biochemical studies, for example, crosslinking experiments and radioligand-binding affinity study. Subsequently, two cDNAs encoding for endothelin receptors were cloned and their amino acid sequences were deduced.1,21 The order of affinity of the three endothelin isopeptides for one receptor type, designated ETA, is endothelin-1≧endothelin-2 ≥ endothelin-3. The other type of receptor, designated ETB, shows equipotent affinity for all three endothelins. These results are consistent with the results of previous pharmacological and biochemical studies and, thus, definite molecular evidence for the existence of two distinct subtypes of endothelin receptors was provided. The existence of more subtypes of endothelin receptor has been a matter of controversy. Much pharmacological evidence has accumulated to suggest that there may exist an ETC receptor specific for endothelin-3, or further sub-subtypes of ETA and ETB receptors. However, there has been no report thus far on the isolation of the cDNA clone encoding for such a subtype or sub-subtype of endothelin receptors from mammalian tissues. One possible explanation is posttranslational modification of ETA or ETB receptor proteins, but to solve such discrepancies, further pharmacological, biochemical, or molecular biological analyses may be necessary.
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In addition to causing constriction of vascular tissue, endothelin isopeptides exert pleiotropic effects on nonvascular tissues, for example, respiratory and gastrointestinal tissues, kidney, endocrine glands, and peripheral and central nervous system. Each endothelin isopeptide has been considered to play specific physiological and/or pathophysiological roles independently in various tissues. The pharmacological effects of endothelin-1 on various tissues and distribution of ETA and ETB receptors is collectively shown in Table 1.
TABLE 1 Distribution of ETA and ETB Receptors Tissue (effect)
ETA
ETB
+++
+
Vascular tissues Arterial smooth muscle (contraction) Venous smooth muscle (contraction)
++
Endothelial cell (release of EDRF)
+++
Cardiac muscle (Positive inotropism)
++
+
(Positive chronotropism)
++
+
Coronary vessel (contraction)
+++
+
++
+
+
+++
Adrenal cortex (release of aldosterone)
+
+++
Anterior pituitary (release of ACTH and prolactin)
+
++
Ovary (stimulation of female hormone synthesis)
++
+
+++
++
Nonvascular smooth muscle (contraction) Airway, gastrointestinal tract, bladder, uterus, etc. Lung (clearance receptor) Other than airways and vascular smooth muscle Endocrine glands
Kidney Afferent and efferent arterioles (contraction) Renal tubule (Na+ excretion and diuresis) Mesangial cell (contraction and proliferation)
+++ +
+++
+
++
Central nervous system Mesencephalon (control of blood pressure) Astrocyte (proliferation)
+++
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BIOLOGICAL ACTIONS WITHIN THE CARDIOVASCULAR SYSTEM Endothelin-1 causes extremely potent and long-lasting vasoconstriction in most mammalian species including humans, both in vivo and in vitro. The in vivo hemodynamic responses to intravenously injected endothelin-1 are complex, depending on the vascular bed, and include both direct vasoconstriction and indirect endothelium-mediated vasodilatation, and reflex-mediated responses. In most arterial and some venous smooth muscle cells, endothelin-1 causes constrictor responses via the stimulation of ETA receptors on the cell membrane. By contrast, endothelin-1 stimulates ETB receptors on endothelial cells and releases EDRF (endothelium-derived relaxing factor: nitric oxide), thereby producing vasodilatation. In cardiac muscle cells, endothelin-1 produces positive inotropic and chronotropic responses via stimulation of mostly ETA receptors. Both ETA and ETB receptors are coupled to various GTP-binding proteins (Gq, Gs, Gi, Go, etc.), suggesting that the downstream signal transduction may differ in various cells depending on the type of GTP-binding proteins coupled. In addition to vasoconstrictor actions (acute effects), endothelin-1 exerts potent mitogenic actions on vascular smooth muscle cells and cardiac myocytes (chronic effects), hence causing vascular and cardiac hypertrophy.18,23,24 These effects are mediated via the stimulation of either ETA or ETB receptors and may be provoked through the sequential activation of intracellular kinase cascade, including raf-1, mitogen-activated protein kinase (MAPK) kinase, MAPK, and S6 kinase.6,7,18
PATHOPHYSIOLOGICAL IMPLICATIONS The development of ETA and/or ETB receptor antagonists and gene-manipulated animals greatly accelerated elucidation of physiological and/or pathophysiological roles of endogenous endothelin-1. In normal states, the release of endothelin-1 seems to be sparse, and it plays no appreciable physiological role in the maintenance of blood pressure. When the production and release of endothelin-1 are increased, endothelin-1 contributes to the pathogenesis of and stimulates the progression of various cardiovascular diseases.11,17,18,20 Although the involvement of endothelin-1 in mild to moderate hypertension remains controversial, it is undoubtedly implicated in certain types of hypertension,12 particularly in malignant stages of the disease. Pulmonary hypertension (PH) is a progressively deteriorating condition characterized by an increase in pulmonary vascular tone and enhanced proliferation of pulmonary vascular smooth muscle cells. There has been no effective therapeutic method for the treatment of this disease. PH is associated with the increase in plasma endothelin-1 levels, which is strongly correlated with the severity of the disease.16–18 These aspects are discussed in more detail in the Respiratory
Chapter | 190 Endothelins
Peptides section of this book. The endothelin receptor antagonists are remarkably efficacious in all animal models thus far investigated.17,18 Bosentan (ETA and ETB receptor antagonist) is now available for the clinical treatment of PH (Fig. 2).2,11 In atheroscelerosis and in various kinds of vascular remodeling, endothelin-1 plays causing or progressing roles in the diseases. Currently, orally active endothelin receptor antagonists, such as bosentan, ambrisentan, macitentan, are applied to cardiovascular patients.2,9,12,22 The concept of the cardiovascular continuum and angiotensin II was put forward by Dzau et al.3–5 As for endothelin, we illustrate the concept of the cardiovascular disease continuum and the target diseases of the endothelin receptor antagonists as in Fig. 3. Endothelin antagonist will be extensively used for cardiac diseases, including cardiac hypertrophy, acute and chronic heart failure.11,18
FIGURE 2 Effects of the ETA/B dual receptor antagonist bosentan treatment on 6-min walking distance. In double-blind, placebo-controlled study, 32 patients with pulmonary hypertension were randomly assigned to receiving bosentan (62.5 mg twice daily) for 4 weeks, then 125 mg twice daily or placebo. From baseline to week 20, bosentan increased the mean 6-min walking distance from 360 m to 437 m, whereas the distance was decreased in the placebo group from 355 m to 340 m. *: P < 0.05 versus baseline, P = 0.021 versus placebo. (From The Lancet 2001;358:1119–1123 (Ref. 2). With permission from Elsevier.)
FIGURE 3 The cardiovascular disease continuum and the target diseases of the endothelin receptor antagonists.
SECTION | XIV Handbook of Biologically Active Peptides: Cardiovascular Peptides
Endogenous endothelin-1 is considered to be involved in the progression of atherosclerosis, hypertension, angina pectoris, myocardial infarction, heart failure, and pulmonary hypertension, and these diseases may become target diseases of the endothelin receptor antagonists (Fig. 3). Endothelin receptor antagonists have been shown to exhibit desirable preventive and/or curative effects in various animal models of these cardiovascular diseases,11,18 suggesting that they are of potential therapeutic value, and some of them are already under clinical use.
REFERENCES 1. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a cDNA encoding and endothelin receptor. Nature 1990;348:730–2. 2. Channick RN, Simonneau G, Sitbon O, Robbins IM, Frost A, Tapson VF, et al. Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet 2001;358(9288):1119–23. 3. Dzau V, Braunwald E. Resolved and unresolved issues in the prevention and treatment of coronary artery disease: a workshop consensus statement. Am Heart J 1991;121:1244–62. 4. Dzau VJ. The cardiovascular continuum and rennin–angiotensin– aldosterone system blockade. J Hypertens Suppl 2005;23(1):S9–17. 5. Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J, et al. The cardiovascular disease continuum validated: clinical evidence of improved patient outcomes: part II: clinical trial evidence (acute coronary syndromes through renal disease) and future directions. Circulation 2006;114(25):2871–91. 6. Goto K, Hama H, Kasuya Y. Molecular pharmacology and pathophysiological significance of endothelin. Jpn J Pharmacol 1996;72:261–90. 7. Goto K. Basic and therapeutic relevance of endothelin-mediated regulations. Biol Pharm Bull 2001;24:1219–30. 8. Hickey KA, Rubanyi GM, Paul RJ, Highsmith RF. Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am J Physiol 1985;252:C550–6. 9. Iglarz M, Binkert C, Morrison K, Fischli W, Gatfield J, Treiber A, et al. Pharmacology of macitentan, an orally active tissue-targeting dual endothelin receptor antagonist. J Pharmacol Exp Ther 2008;327(3):736–45. 10. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, et al. The human endothelin family. Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A 1989;86:2863–7. 11. Khimiji Al-K, Rockey DC. Endothelin-biology and disease. Cell Signal 2010;22:1615–25. 12. Krum H, Viskoper RJ, Lacourciere Y, Budde M, Charlon V. The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. N Engl J Med 1998;338:784–90.
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13. Maeda S, Miyauchi T, Goto K, Matsuda M. Differences in the change in the time course of plasma endothelin-1 and endothelin-3 levels after exercise in humans: the response to exercise of endothelin-3 is more rapid than that of endothelin-1. Life Sci 1997;61:419–25. 14. Maeda S, Miyauchi T, Kakiyama T, Sugawara J, Iemitsu M, IrukayamaTomobe Y, et al. Effects of exercise training of 8 weeks and detraining on plasma levels of endothelium-derived factors, endothelin-1 and nitric oxide, in healthy young humans. Life Sci 2001;69:1005–16. 15. Maeda S, Tanabe T, Miyauchi T, Otsuki T, Sugawara J, Iemitsu M, et al. Aerobic exercise training reduces plasma endothelin-1 concentration in older women. J Appl Physiol 2003;95:336–41. 16. Miyauchi T, Yanagisawa M, Tomizawa T, Sugishita Y, Suzuki N, Fujino M, et al. Increased plasma concentrations of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet 1989;ii(8653):53–4. 17. Miyauchi T, Yorikane R, Sakai S, Sakurai T, Okada M, Nishikibe M, et al. Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rat with monocrotaline-induced pulmonary hypertension. Circ Res 1993;73:887–97. 18. Miyauchi T, Masaki T. Pathophysiology of endothelin in the cardiovascular system. Annu Rev Physiol 1999;61:391–415. 19. O’Brien RF, Robbins RJ, McMurthy IF. Endothelial cells in culture produce a vasoconstrictor substance. J Cell Physiol 1987;132:263–70. 20. Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, Sugishita Y. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure. Nature 1996;384:353–5. 21. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 1990;348:732–5. 22. Sidharta PN, van, Giersbergen PL, Halabi A, Dingemanse J. Macitentan: entry-into-humans study with a new endothelin receptor antagonist. Eur J Clin Pharmacol 2011;67(10):977–84. 23. Shimojo N, Jesmin S, Zaedi S, Maeda S, Soms M, Aonuma K, et al. Eicosapentaenoic acid prevents endothelin-1-induced cardiomyocyte hypertrophy in vitro through the suppression of TGF-beta 1 and phosphorylated JNK. Am J Physiol Heart Circ Physiol 2006;291:H835–45. 24. Shimojo N, Jesmin S, Zaedi S, Otsuki T, Maeda S, Yamaguchi N, et al. Contributory role of VEGF overexpression in endothelin-1-induced cardiomyocyte hypertrophy. Am J Physiol Heart Circ Physiol 2007;293:H474–81. 25. Suzuki N, Matsumoto H, Kitada C, Masaki T, Fujino M. A sensitive sandwich-enzyme immunoassay to detect immunoreactive big endothelin in plasma. J Immunol Methods 1989;118:245–50. 26. Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, deWit D, Yanagisawa M. ECE-1: A membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothein-1. Cell 1994;78:473–85. 27. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–5.
Endothelins Takashi Miyauchi and Katsutoshi Goto
ABSTRACT A potent vasoconstrictor peptide, endothelin (endothelin-1), was discovered from culture supernatant of porcine endothelial cells in 1988. From human genomic DNA analysis, two other family peptides, endothelin-2 and endothelin-3, were found. They showed different effects and distribution, suggesting that each peptide may play separate roles in different organs. Endothelins act via the activation of two receptor subtypes, ETA and ETB receptors, both of which are coupled to various GTPbinding proteins depending on cell types. Endogenous ET-1 may be involved in the progression of various cardiovascular diseases, and ET antagonists are currently used clinically in the treatment of patients with pulmonary hypertension and are considered to have further target diseases such as systemic hypertension, heart failure, cardiac diseases, renal diseases.
DISCOVERY Vasoconstriction dependent on or enhanced by intact endothelium has been observed in response to various chemical and physical stimuli such as norepinephrine, thrombin, hypoxia, increased transmural pressure, mechanical stretch. These observations lead to the speculation that endothelial cells may release certain vascular constricting substance(s), endothelium-derived contracting factors (EDCF). In 1985, Hickey et al. attempted to test the biological activity of the culture medium of bovine aortic endothelial cells on isolated porcine coronary arteries, and they found that the culture supernatant contained peptide-like factor(s), which triggered a slowly developing and long-lasting contraction of the coronary arteries. Based on gel chromatography analysis, they suggested that this particular EDCF is a peptide with a molecular mass of approximately 8500 Da.8 At about the same time, Gillespie et al. also detected vascular constricting activity in the culture supernatant of porcine aortic endothelial cells. They subsequently reported that the activity was increased when the cultured endothelial cells were stimulated by thrombin, and they suggested that this EDCF is a peptide with a molecular mass of approximately 3000 Da.19 1402
In 1987, we performed experiments similar to those of Hickey et al. and confirmed that the supernatant from confluent monolayer cultures of porcine aortic endothelial cells contained a slowly developing and long-lasting vascular constricting factor(s), peptidic in nature, because the vascular constricting activity was abolished by pretreatment of the conditioned medium with trypsin. The activity was also detected in serum-free conditioned medium, and no appreciable change in activity was observed even after long-term (2–3 weeks) maintenance of the endothelial cell culture in serum-free condition. The successful attempt at serum-free maintenance and detection of vascular constricting activity prompted us to isolate and purify the active peptide in the supernatant because of the absence of interference with proteins and/or peptides in the serum itself. Cells isolated from porcine thoracic aortas and grown to a confluent monolayer were maintained in serum-free minimum essential medium. The medium was changed every 5 days, and the conditioned medium was pooled at −20 °C. The pooled conditioned medium was first centrifuged at 1000 g for 20 min, and subsequently, the supernatant was desalted and concentrated. The concentrated medium was loaded onto an anion-exchange column and eluted by applying a linear gradient of NaCl. The vascular constricting activity of the eluent was assayed by adding a small amount of each fraction directly into a muscle chamber where a helical strip of porcine coronary artery with the intima denuded was suspended, and the active fraction was collected. The active fraction was subjected to reversedphase high-performance liquid chromatography (HPLC) and elution with a linear gradient of acetonitrile, and the vascular constricting activity of each fraction was similarly assayed. A second trial of reversed-phase HPLC enabled us to purify the active component. Approximately 3 nmol of the final product was obtained, which was just enough to perform subsequent amino acid analysis. The purified peptide was subjected to amino acid sequence analysis by means of an automated gas-phase peptide sequencer and carboxy-terminal analysis by hydrazinolysis (Edman’s reaction). As a result, the peptide was revealed to be composed of 21 amino acid residues with Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00190-1 Copyright © 2013 Elsevier Inc. All rights reserved.
SECTION | XIV Handbook of Biologically Active Peptides: Cardiovascular Peptides
free amino- and carboxy-termini. At first, the carboxyterminal amino acid (tryptophan) was not detected, and a 20 amino acid peptide synthesized artificially did not exhibit any biological activity. It was soon recognized that the structure of tryptophan (indole ring) is easily degraded during Edman’s reaction. The peptide comprising 21 amino acid residues subsequently synthesized did exhibit vascular constricting activity identical to that of natural peptide. The four cysteine residues at the amino-terminal portion were found to form two intramolecular disulfide bonds, the topological analysis of which revealed that the arrangement of disulfide bonds is a coaxial form (1–15 and 3–11). Because it was originally discovered from the culture supernatant of endothelial cells, the peptide was termed “endothelin.”27
STRUCTURE OF THE mRNA/GENE Based on the amino acid residues 7–20 of endothelin, several kinds of oligonucleotides were synthesized according to the mammalian codon usage statistics. Using one of the synthesized oligonucleotides as a single “optimal” DNA probe, approximately 2 × 106 clones were screened from aλgt10 cDNA library constructed for porcine endothelial cell mRNA, and 38 hybridization-positive clones were soon identified. Four of these clones were subjected to further characterization, and finally, a complete nucleotide sequence of porcine preproendothelin cDNA and the deduced amino acid sequence were determined.
PROCESSING OF THE PRECURSOR The 203 residue porcine preproendothelin contains 19 residues of deduced N-terminal amino acid sequence, being characteristic of a secretory signal sequence, that is, a hydrophobic core followed by residues with small polar side chains. As anticipated, paired basic amino acid residues Lys51-Arg52, which are recognized by the usual processing endopeptidase, furin (see the furin chapter in Peptide Biosynthesis/Processing section of the book), directly precede the endothelin sequence, but no dibasic pair is found thereafter until Ara92-Arg93. This indicates that the intermediate peptide of 39 amino acids, called big endothelin (Cys53Arg92), may be first generated, and subsequently, mature endothelin may be generated via the unusual proteolytic processing between Try73 and Val74 in big endothelin by an endopeptidase exhibiting chymotrypsin-like specificity, which was putatively termed “endothelin-converting enzyme (ECE).” This presumptive pathway of endothelin biosynthesis in endothelial cells was confirmed later. The human preproendothelin cDNA was soon cloned, and its sequence of 212 amino acid residues containing big endothelin of 38 amino acid sequences was determined (Fig. 1). The cDNA of ECE was also cloned, and it was revealed that ECE is a 785 amino acid metalloprotease containing a
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single membrane-spanning sequence with only a 56-residue N-terminal cytoplasmic tail and an extracellular C-terminal of 681 amino acid residues that contains the catalytic domain.26 Amino acid residues 593–601 match the highly conserved consensus sequence of a zinc-binding motif, HEXXH, which is shared by many known metalloproteases. The ECE protein has ten predicted sites for N-glycosylation in the extracellular domain, suggesting that ECE behaves as a highly glycosylated protein.
DISTRIBUTION OF THE mRNA/PEPTIDE IN THE CARDIOVASCULAR SYSTEM Southern blot analysis of human genomic DNA under low hybridization stringency with a 42-mer synthetic oligonucleotide probe corresponding to amino acid residues 7–20 of endothelin, showed that three different restriction fragments were always detected regardless of the restriction endonucleases used. The nucleotide sequences encoding amino acid residues of the three endothelins are highly conserved among the three genes, with 77–82% of the nucleotide residues being identical. By contrast, the nucleotide sequences upstream from the mature peptides are very poorly conserved. These observations suggest that although the three genes are evolutionally relatively distant from each other, the genes evolved from a common ancestral gene under strong pressure to preserve mature endothelin sequences. The three peptides were designated endothelin-1, endothelin-2, and endothelin-3.10 Endothelin-1 is the original peptide corresponding to that detected in the culture medium of porcine aortic endothelial cells. Although vascular endothelial cells are the major source of endothelin-1, Northern blot analysis revealed that the genes encoding the three endothelin isopeptides are expressed with different patterns in a wide variety of cell types including cardiac myocytes, vascular smooth muscle cells, pituitary cells, macrophages, and mast cells, suggesting that the peptides may participate independently in complex regulatory mechanisms in various organs. The conservation of numerous endothelin-related genes was also observed in several mammalian species examined, including humans, pigs, and rats. Further, four cardiotoxic peptides highly homologous to endothelins, sarafotoxins (S6a–S6d), were isolated from the snake venom of Atractaspis engaddensis. Endothelin-like immunoreactivity was also found in several species of invertebrates and fishes, indicating that endothelins found in humans seem to have a long evolutionary history. Since endothelin-1 was discovered first and showed a wide variety of actions not only on the cardiovascular system but also on various other tissues, much information has accumulated on endothelin-1 compared with the two other peptides. The method for measurement of endothelin-1 levels in plasma or various tissues by means of
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Chapter | 190 Endothelins
FIGURE 1 The biosynthesis, intracellular processing of endothelin-1 (ET-1) from preproET-1. Endothelin-converting enzyme (ECE) specifically catalyzes the conversion of big ET-1 to ET-1. Cys–Cys represents the intramolecular disulfide bond.
radioimmunoassay (RIA) or enzyme immunoassay (EIA)25 was established soon after the discovery of endothelin-1. RIA uses only one kind of antibody against endothelin-1, whereas EIA uses two kinds of antibodies, each recognizing the N-terminal and C-terminal portions of endothelin-1, respectively. At an early stage, the reported plasma concentrations of endothelin-1 in humans were fairly divergent, ranging from 0.2 to 18.5pg/ml in healthy subjects. To avoid unnecessary confusion, the reason for the difference in the values of plasma endothelin-1 level was examined thoroughly and, as a result, it was generally agreed that the divergence originated mostly from differences in the specificity of the antibodies used and in the recovery rate during extraction of endothelin-1 from plasma or tissues. It is now accepted that the plasma concentration of endothelin-1 in healthy subjects is 1.0–2.0pg/ml,13–16 which is at least one order of magnitude lower than that of circulating human natriuretic peptides and several times less than that
of angiotensin II. Plasma concentrations of endothelin-1 increase drastically in the case of various cardiovascular diseases.13–16 Endothelin-1 may be released in both luminal and abluminal directions from endothelial cells in vivo. Luminal released endothelin-1 may be diluted by the blood stream, and its circulating concentration (1.0–2.0pg/ml) is below the threshold concentration producing vasoconstriction. Although the exact concentration in the abluminal space (vascular smooth muscle surface) is not known, endothelin-1 is more likely to be a locally acting rather than a circulating hormone. When endothelin-1 production and secretion are increased drastically in certain sites under pathophysiological conditions, severe local vasoconstriction might be anticipated. When endothelin-1 is intravenously administered as a bolus, it disappears quite rapidly from the blood stream with a half-life of a few minutes. This rapid removal of endothelin-1 from the circulation results from uptake into
SECTION | XIV Handbook of Biologically Active Peptides: Cardiovascular Peptides
various tissues, including the lung, kidney, spleen, and liver. The lung seems to be one of the most important tissues for uptake, because approximately 60% of endothelin-1 is removed after a single passage through the pulmonary circulation. In lung tissues, the ETB receptor is highly expressed and endothelin-1-ETB receptor complex may be taken up into cells through an internalization process and degraded by lysozomal enzymes, for example, aspartic proteases. Neutral endopeptidase and lysozomal cathepsin G are also concerned with enzymatic degradation of endothelin-1. Despite such a rapid disappearance from the circulation, the blood pressure–elevating effect of endothelin-1 continues for an extremely long period; stabilization of the endothelin-1-ETA receptor complex may contribute, but the exact reason for this phenomenon is unknown.
RECEPTORS AND THEIR DISTRIBUTION Endothelin-induced responses can be divided into two major groups, according to the pharmacological potency order of the three isopeptides. In the first group of responses, including vascular constriction, broncho-constriction, uterine smooth muscle constriction, and stimulation of aldosterone secretion, endothelin-1 and endothelin-2 act as far more potent agonists than endothelin-3 do. In the second group, which includes endothelium-dependent vascular relaxation, astrocyte proliferation, and inhibition of ex vivo platelet aggregation, the three isopeptides possess almost equal potency. These findings suggest that there are at least two distinct endothelin receptors mediating these two distinct groups of pharmacological responses. The existence of multiple receptors was also supported by biochemical studies, for example, crosslinking experiments and radioligand-binding affinity study. Subsequently, two cDNAs encoding for endothelin receptors were cloned and their amino acid sequences were deduced.1,21 The order of affinity of the three endothelin isopeptides for one receptor type, designated ETA, is endothelin-1≧endothelin-2 ≥ endothelin-3. The other type of receptor, designated ETB, shows equipotent affinity for all three endothelins. These results are consistent with the results of previous pharmacological and biochemical studies and, thus, definite molecular evidence for the existence of two distinct subtypes of endothelin receptors was provided. The existence of more subtypes of endothelin receptor has been a matter of controversy. Much pharmacological evidence has accumulated to suggest that there may exist an ETC receptor specific for endothelin-3, or further sub-subtypes of ETA and ETB receptors. However, there has been no report thus far on the isolation of the cDNA clone encoding for such a subtype or sub-subtype of endothelin receptors from mammalian tissues. One possible explanation is posttranslational modification of ETA or ETB receptor proteins, but to solve such discrepancies, further pharmacological, biochemical, or molecular biological analyses may be necessary.
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In addition to causing constriction of vascular tissue, endothelin isopeptides exert pleiotropic effects on nonvascular tissues, for example, respiratory and gastrointestinal tissues, kidney, endocrine glands, and peripheral and central nervous system. Each endothelin isopeptide has been considered to play specific physiological and/or pathophysiological roles independently in various tissues. The pharmacological effects of endothelin-1 on various tissues and distribution of ETA and ETB receptors is collectively shown in Table 1.
TABLE 1 Distribution of ETA and ETB Receptors Tissue (effect)
ETA
ETB
+++
+
Vascular tissues Arterial smooth muscle (contraction) Venous smooth muscle (contraction)
++
Endothelial cell (release of EDRF)
+++
Cardiac muscle (Positive inotropism)
++
+
(Positive chronotropism)
++
+
Coronary vessel (contraction)
+++
+
++
+
+
+++
Adrenal cortex (release of aldosterone)
+
+++
Anterior pituitary (release of ACTH and prolactin)
+
++
Ovary (stimulation of female hormone synthesis)
++
+
+++
++
Nonvascular smooth muscle (contraction) Airway, gastrointestinal tract, bladder, uterus, etc. Lung (clearance receptor) Other than airways and vascular smooth muscle Endocrine glands
Kidney Afferent and efferent arterioles (contraction) Renal tubule (Na+ excretion and diuresis) Mesangial cell (contraction and proliferation)
+++ +
+++
+
++
Central nervous system Mesencephalon (control of blood pressure) Astrocyte (proliferation)
+++
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BIOLOGICAL ACTIONS WITHIN THE CARDIOVASCULAR SYSTEM Endothelin-1 causes extremely potent and long-lasting vasoconstriction in most mammalian species including humans, both in vivo and in vitro. The in vivo hemodynamic responses to intravenously injected endothelin-1 are complex, depending on the vascular bed, and include both direct vasoconstriction and indirect endothelium-mediated vasodilatation, and reflex-mediated responses. In most arterial and some venous smooth muscle cells, endothelin-1 causes constrictor responses via the stimulation of ETA receptors on the cell membrane. By contrast, endothelin-1 stimulates ETB receptors on endothelial cells and releases EDRF (endothelium-derived relaxing factor: nitric oxide), thereby producing vasodilatation. In cardiac muscle cells, endothelin-1 produces positive inotropic and chronotropic responses via stimulation of mostly ETA receptors. Both ETA and ETB receptors are coupled to various GTP-binding proteins (Gq, Gs, Gi, Go, etc.), suggesting that the downstream signal transduction may differ in various cells depending on the type of GTP-binding proteins coupled. In addition to vasoconstrictor actions (acute effects), endothelin-1 exerts potent mitogenic actions on vascular smooth muscle cells and cardiac myocytes (chronic effects), hence causing vascular and cardiac hypertrophy.18,23,24 These effects are mediated via the stimulation of either ETA or ETB receptors and may be provoked through the sequential activation of intracellular kinase cascade, including raf-1, mitogen-activated protein kinase (MAPK) kinase, MAPK, and S6 kinase.6,7,18
PATHOPHYSIOLOGICAL IMPLICATIONS The development of ETA and/or ETB receptor antagonists and gene-manipulated animals greatly accelerated elucidation of physiological and/or pathophysiological roles of endogenous endothelin-1. In normal states, the release of endothelin-1 seems to be sparse, and it plays no appreciable physiological role in the maintenance of blood pressure. When the production and release of endothelin-1 are increased, endothelin-1 contributes to the pathogenesis of and stimulates the progression of various cardiovascular diseases.11,17,18,20 Although the involvement of endothelin-1 in mild to moderate hypertension remains controversial, it is undoubtedly implicated in certain types of hypertension,12 particularly in malignant stages of the disease. Pulmonary hypertension (PH) is a progressively deteriorating condition characterized by an increase in pulmonary vascular tone and enhanced proliferation of pulmonary vascular smooth muscle cells. There has been no effective therapeutic method for the treatment of this disease. PH is associated with the increase in plasma endothelin-1 levels, which is strongly correlated with the severity of the disease.16–18 These aspects are discussed in more detail in the Respiratory
Chapter | 190 Endothelins
Peptides section of this book. The endothelin receptor antagonists are remarkably efficacious in all animal models thus far investigated.17,18 Bosentan (ETA and ETB receptor antagonist) is now available for the clinical treatment of PH (Fig. 2).2,11 In atheroscelerosis and in various kinds of vascular remodeling, endothelin-1 plays causing or progressing roles in the diseases. Currently, orally active endothelin receptor antagonists, such as bosentan, ambrisentan, macitentan, are applied to cardiovascular patients.2,9,12,22 The concept of the cardiovascular continuum and angiotensin II was put forward by Dzau et al.3–5 As for endothelin, we illustrate the concept of the cardiovascular disease continuum and the target diseases of the endothelin receptor antagonists as in Fig. 3. Endothelin antagonist will be extensively used for cardiac diseases, including cardiac hypertrophy, acute and chronic heart failure.11,18
FIGURE 2 Effects of the ETA/B dual receptor antagonist bosentan treatment on 6-min walking distance. In double-blind, placebo-controlled study, 32 patients with pulmonary hypertension were randomly assigned to receiving bosentan (62.5 mg twice daily) for 4 weeks, then 125 mg twice daily or placebo. From baseline to week 20, bosentan increased the mean 6-min walking distance from 360 m to 437 m, whereas the distance was decreased in the placebo group from 355 m to 340 m. *: P < 0.05 versus baseline, P = 0.021 versus placebo. (From The Lancet 2001;358:1119–1123 (Ref. 2). With permission from Elsevier.)
FIGURE 3 The cardiovascular disease continuum and the target diseases of the endothelin receptor antagonists.
SECTION | XIV Handbook of Biologically Active Peptides: Cardiovascular Peptides
Endogenous endothelin-1 is considered to be involved in the progression of atherosclerosis, hypertension, angina pectoris, myocardial infarction, heart failure, and pulmonary hypertension, and these diseases may become target diseases of the endothelin receptor antagonists (Fig. 3). Endothelin receptor antagonists have been shown to exhibit desirable preventive and/or curative effects in various animal models of these cardiovascular diseases,11,18 suggesting that they are of potential therapeutic value, and some of them are already under clinical use.
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13. Maeda S, Miyauchi T, Goto K, Matsuda M. Differences in the change in the time course of plasma endothelin-1 and endothelin-3 levels after exercise in humans: the response to exercise of endothelin-3 is more rapid than that of endothelin-1. Life Sci 1997;61:419–25. 14. Maeda S, Miyauchi T, Kakiyama T, Sugawara J, Iemitsu M, IrukayamaTomobe Y, et al. Effects of exercise training of 8 weeks and detraining on plasma levels of endothelium-derived factors, endothelin-1 and nitric oxide, in healthy young humans. Life Sci 2001;69:1005–16. 15. Maeda S, Tanabe T, Miyauchi T, Otsuki T, Sugawara J, Iemitsu M, et al. Aerobic exercise training reduces plasma endothelin-1 concentration in older women. J Appl Physiol 2003;95:336–41. 16. Miyauchi T, Yanagisawa M, Tomizawa T, Sugishita Y, Suzuki N, Fujino M, et al. Increased plasma concentrations of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet 1989;ii(8653):53–4. 17. Miyauchi T, Yorikane R, Sakai S, Sakurai T, Okada M, Nishikibe M, et al. Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rat with monocrotaline-induced pulmonary hypertension. Circ Res 1993;73:887–97. 18. Miyauchi T, Masaki T. Pathophysiology of endothelin in the cardiovascular system. Annu Rev Physiol 1999;61:391–415. 19. O’Brien RF, Robbins RJ, McMurthy IF. Endothelial cells in culture produce a vasoconstrictor substance. J Cell Physiol 1987;132:263–70. 20. Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, Sugishita Y. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure. Nature 1996;384:353–5. 21. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 1990;348:732–5. 22. Sidharta PN, van, Giersbergen PL, Halabi A, Dingemanse J. Macitentan: entry-into-humans study with a new endothelin receptor antagonist. Eur J Clin Pharmacol 2011;67(10):977–84. 23. Shimojo N, Jesmin S, Zaedi S, Maeda S, Soms M, Aonuma K, et al. Eicosapentaenoic acid prevents endothelin-1-induced cardiomyocyte hypertrophy in vitro through the suppression of TGF-beta 1 and phosphorylated JNK. Am J Physiol Heart Circ Physiol 2006;291:H835–45. 24. Shimojo N, Jesmin S, Zaedi S, Otsuki T, Maeda S, Yamaguchi N, et al. Contributory role of VEGF overexpression in endothelin-1-induced cardiomyocyte hypertrophy. Am J Physiol Heart Circ Physiol 2007;293:H474–81. 25. Suzuki N, Matsumoto H, Kitada C, Masaki T, Fujino M. A sensitive sandwich-enzyme immunoassay to detect immunoreactive big endothelin in plasma. J Immunol Methods 1989;118:245–50. 26. Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, deWit D, Yanagisawa M. ECE-1: A membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothein-1. Cell 1994;78:473–85. 27. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–5.