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Endothelins
CHAPTER 36
Endothelins Hirokazu Ohtaki
Abbreviation: ET, EDN Endothelin (ET), also called ET-1, was first isolated from culture supernatant of porcine endothelial cells. The hormone shows a potent vasoconstructive activity. There are three isopeptides, ET-1, -2 and -3.
Discovery Presence of a potent vasoconstructor in the supernatant of porcine endothelial cells was reported and isolated as ET-1 in 1988 [1].
Structure Structural Features ET-1 consists of 21 aa residues with free amino- and carboxyl-termini including a hydrophobic C-terminus and four cysteine residues which form two disulfide bonds (Figure 36.1). There are two isopeptides, ET-2, and ET-3, differing by the 2nd and 6th amino acids, respectively. ET-1 is the most abundant isoform.
Primary Structure Mature ET-1 is conserved (only one aa difference) in fish and the higher organisms.
Properties Mr of porcine ET-1 is 2,492 and predicted pI is 9.5. Soluble in water. ET-1 solution in water at 1 mg/ml is stable for more than a year at 220 C.
Synthesis and Release Gene and mRNA In the human, the ET-1 (EDN1), ET-2 (EDN2), and ET-3 (EDN3) genes are located on chromosome-6 (6p24.1), -1 (1p34), and -20 (20q13.2-q13.3). Human ET-1, ET-2 and ET-3 mRNA have 2109, 1258, and 2438 bp respectively. Genetic fine mapping studies of human EDN1 localized it to the telomeric region of chromosome 6p, close to the gene encoding the α-subunit of clotting factor XIII. The human EDN1 contains five exons, four introns, and 50 - and 30 -flanking regions and spans approximately 6.8 kb of DNA [2]. The ET-1 mRNA is 2.3 kb in length and directs translation of the precursor prepro-ET-1 peptide, including the sequence of pro-ET-1 (big ET) and the ET-like peptide (ETLP). The ET-1 promoter contains two functional transcription start sites including 326
TATAA and CAAT boxes, nuclear factor -1 (NF-1), GATA-2, AP-1/jun, and acute phase reaction regulatory elements (APRE).
Precursors All three ET precursors are processed by two proteases to digest the mature active forms [3]. For ET-1, the 212-residue preproendothelin, including 19 residues of signal sequence which is characteristic of a secretary signal sequence, are cleaved at the dibasic site by fulin-like endopoptidase to produce big endothelins (big ETs). The big ETs consist of 37 to 41 aa residues and are an inactive intermediate form. Mature and active ET-1 is formed by a family of membrane-bound zinc metalloproteinases, from the neprilysin superfamily, termed endothelin-converting enzyme (ECEs) [4]. Other enzymes such as non-ECE metalloproteinase, cathepsin A, and chymase contribute to the degradation.
Tissue Distribution of mRNA ET-1 is most abundantly localized in vascular endothelial cells, including umbilical vein, mesenteric artery, glomerulus, corpus cavernosum, aorta, and brain microvessels [1,2]. However, ET isopeptides are expressed in a variety of tissues and cell types and the isopeptides express a tissue-specific pattern. ET-1 mRNA expression is observed in breast epithelium, keratinocytes, endometrial stromal and glandular epithelial cells, macrophages, bone marrow mast cells, astrocytes, mesangial cells, neurons of the spinal cord and dorsal root ganglia, avascular human amnion, cardiomyocytes, and some tumor-derived cells. Few data are available for ET-2 and ET-3 in specific cell types, although a human renal adenocarcinoma cell line was shown to express ET-2. At the tissue level of rat, ET-1 expresses in vascular endothelium, lung, brain, uterus, stomach, heart, adrenal gland, and kidney [5]. ET-3 mRNA expresses in eyeball, submandibular gland, brain, kidney, jejunum, stomach, and spleen with northern blotting. Although ET-2 mRNA expresses in large and small intestine, skeletal muscle, heart, and stomach, large and small intestine expression is greatest.
Tissue and Plasma Concentrations Plasma level of ET-1 in healthy human is B2.0 pg/ml by specific radioimmunoassay (RIA). Other studies of ET-1 levels with RIA demonstrated in rat that the inner medulla of the kidney had the highest concentration (8.7 6 2.2 pg/mg of wet weight).
Y. Takei, H. Ando, & K. Tsutsui (Eds): Handbook of Hormones. DOI: http://dx.doi.org/10.1016/B978-0-12-801028-0.00036-2 © 2016 Elsevier Inc. All rights reserved.
C H A P T E R 3 6 Endothelins Biological Functions Target Cells/Tissues and Functions ET-1 causes strong and lasting vasoconstriction and raises blood pressure. Therefore, ET-1 was expected to contribute to the functioning of the cardiovascular system. Moreover, in addition to vasoconstriction, ET-1 acts on various cardiovascular cells directly or indirectly causing cell proliferation and production of diverse active substances, including extracellular matrix. ET-1 is involved in renal diseases, cancer, diabetes and insulin resistance, allograft rejection, and renal diseases, as well as cardiovascular diseases. Figure 36.1 Primary structure of mammal endothelins. Ser4 is substituted with Asn4 in mouse.
Regulation of Synthesis and Release ET-1 is transcriptionally regulated by stimuli such as shear stress, hypoxia, cytokines, lipopolysaccharide, and growth factors. These enhance ET-1 mRNA transcription and protein secretion; ET-1 shows autocrine activity. Some of them may contribute to the stability of preproET-1 mRNA.
Receptors Structure and Subtype Two G protein-coupled 7-transmembrane receptors (ETA and ETB) have been identified in humans. Both receptor types contain 7-transmembrane domain 2226 hydrophobic aa in their B400-aa sequences. The ETA exhibits subnanomolar affinities for ET-1 and ET-2 and 100-fold lower affinity for ET3 [3]. The ETB receptor has equal subnanomolar affinities for all ETs. The ETA is considered the primary vasoconstrictor and growth-promoting receptor, and the ETB inhibits cell growth and vasoconstriction in the vascular system. The ETB also functions as a “clearance receptor” which metabolites ET-1. This clearance mechanism is particularly important in the lung with metabolites being about 80% of ET-1 in the circulation.
Signal Transduction Pathway After ET-1 binding to ETA on smooth muscle cells, PLC is activated via G-coupled protein and produces IP3 and DG. IP3 induces the release of Ca21 in endoplasmic reticulum. Activation of ETA also induces the influx of extracellular Ca21 via the voltage-dependent Ca21 channel, receptor-operated Ca21 channel, and nonselective cation channel. The Ca21 influx increases [Ca21]i level and finally induces the phosphorylation of the myosin light chain and leads to contraction of the smooth muscle cells.
Agonist No synthetic agonist is shown for ETA. Agonists for ETB are {Ala1,3,11,15} ET-1, BQ3020, IRL-1620, and S6c.
Antagonist Peptide and non-peptide ET receptor selective or nonselective antagonists have been developed by pharmaceutical companies. Ro47-0203 (Bosentan) is a non-selective ET receptor antagonist used in the treatment of pulmonary artery hypertension (PAH). Representative selective ETA receptor antagonists are BQ-123, BMS182874, LU135252 (darusentan) and PD156707. Representative selective ETB receptor antagonists are BQ-788 and RES701-1. Representative non-selective ETA/ETB receptor antagonists are TAK-044 and Ro47-0203 (bosentan).
Phenotype in Gene-Modified Animals A series of gene-deficient mice of ETs and the receptors are reported from the Yanagisawa group. The ligand or receptor gene-deficient mice showed several developmental abnormalities. Homozygous ET-1 knockout mice have craniofacial and cardiac malformations that lead to neonatal death [6]. ET-2 knockout mice exhibit growth retardation, hypothermia, hypoxemic hypoxia, hypercapnia, emphysema, and premature death [7]. ET-3 knockout mice exhibit aganglionic megacolon with white spotting of the hair coat due to impaired expansion and differentiation of epidermal melanoblasts. Mutants die around weaning with impacted colons [8]. Homozygous inactivation of ETA causes perinatal lethality and craniofacial deformities including middle ear defects. The knockout mice also show severe cardiac outflow and great vessel anomalies, respiratory and PNS defects, thymus and tongue hypoplasia, and absent salivary glands [9]. ETB knockout mice show pigmentation limited to small patches on the head and rump, exhibit abnormal neural epithelium of the inner ear, and die from megacolon resulting from impaired neuronal migration and aganglia [10].
Pathophysiological Implications Clinical Implications Plasma ET1 level was measured in diverse abnormal states of humans with RIA and was reported to increase in abnormal states such as pulmonary hypertension, acute myocardial infarction, essential hypertension, subarachnoid hemorrhage, and diabetes mellitus. The plasma level of ET-1 increases with the severity of congestive heart failure (CHF) and, in particular, increases in patients nominated as Class IV of the New York Heart Association (NYHA) functional classification. The plasma ET-1 and big-ET-1 levels are inversely correlated with the left ventricle ejection fraction (LVEF) and are independent predictors of survival in patients with severe CHF.
Use for Diagnosis and Treatment ET-1 concentration is not measured to diagnosis in clinical settings. A non-selective ETA/ETB antagonist (bosentan; Tracleers) is used for treatment of pulmonary hypertension. A selective ETA antagonist (ambrisentan; Volibriss) has also been used for the disease since 2010. References 1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411415. 2. Rubanyi GM, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev. 1994;46:325415. 3. Kedzierski RM, Yanagisawa M. Endothelin system: the doubleedged sword in health and disease. Annu Rev Pharmacol Toxicol. 2001;41:851876.
327
P A R T I Peptides and Proteins in Vertebrates 4. Barton M, Yanagisawa M. Endothelin: 20 years from discovery to therapy. Can J Physiol Pharmacol. 2008;86:485498. 5. Sakurai T, Masaki T. Endothelin receptors—an overview. Tanpakushitsu Kakusan Koso. 1991;36:23812388. 6. Kurihara Y, Kurihara H, Suzuki H, et al. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature. 1994;368:703710. 7. Chang I, Bramall AN, Baynash AG, et al. Endothelin-2 deficiency causes growth retardation, hypothermia, and emphysema in mice. J Clin Invest. 2013;123:26432653.
8. Baynash AG, Hosoda K, Giaid A, et al. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell. 1994;79:12771285. 9. Clouthier DE, Hosoda K, Richardson JA, et al. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development. 1998;125:813824. 10. Hosoda K, Hammer RE, Richardson JA, et al. Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell. 1994;79:12671276.
Supplemental Information
E-Figure 36.1 Human endothelin-1 (EDN1) gene located on chromosome 6 (6p24.1).
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C H A P T E R 3 6 Endothelins
E-Figure 36.2 The alignment of amino acid sequence of the EDN1 in vertebrates. Conserved amino acid residues are indicated in green, and Cys residues are indicated in yellow. Mature endothelin-1 is conserved in (only one aa difference) in fish and the higher organisms. Accession numbers: human, NP_001161791; chimpanzee, XP_518241; monkey, XP_001089874; dog, NP_001002956; cow, NP_851353; mouse, NP_034234; rat, NP_036680; chicken, XP_418943; frog, XP_002932710; zebrafish, NP_571594.
E-Figure 36.3 Human endothelin-1 receptor type A (EDNRA) gene located on chromosome 4 (4q31.22).
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P A R T I Peptides and Proteins in Vertebrates
E-Figure 36.4 Human endothelin-1 receptor type B (EDNRB) gene located on chromosome 13 (13q22).
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C H A P T E R 3 6 Endothelins
E-Figure 36.5 The alignment of amino acid sequence of the EDNRA in vertebrates. Conserved amino acid residues are indicated in green, and Cys residues are indicated in yellow. Accession numbers: human, NP_001948; chimpanzee, XP_001149597; monkey, NP_001248421, XP_001098827; dog, NP_001026802 XP_539756; cow, NP_776733; mouse, NP_034462, XP_134499; rat, NP_036682, XP_001054233; chicken, NP_989450; frog, NP_001072275; zebrafish, NP_001092915, XP_687609.
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P A R T I Peptides and Proteins in Vertebrates
E-Figure 36.6 The alignment of amino acid sequence of the EDNRB in vertebrates. Conserved amino acid residues are indicated in green, and Cys residues are indicated in yellow. Accession numbers: human, NP_001188326; chimpanzee, XP_509693; monkey, XP_001089047; dog, NP_001010943; cow, NP_776734; mouse, NP_001129533; rat, NP_059029, XP_344452; chicken; NP_001001127, XP_417001; frog, NP_001072476; zebrafish, NP_571272.
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C H A P T E R 3 6 Endothelins E-Table 36.1 Lists of Agonists and Antagonists for ETRs Selective ETA antagonist peptide
BQ-123 BQ-610 FR139317 TTA 788 nonpeptide ABT-627 (arasentan) BMS182874 LU135252 (darusentan) PD156707 PD176856 Ro61-612 (tezosentan) Ro61-1790 S-0139 EMD122946 SB247083 TBC11251 (sitaxsentan) ZD1611 T-0201 J-104121 J-104131 YM598 N/A
agonist
Selective ETB
Nonselective ETA/ETB
BQ-788 IRL2500 RES701-1
PD142893 TAK044
A-192621
A-182086
K-8794
L-754142 Ro46-2005 Ro47-0203 (bosentan) SB209670 SB217242 (enrasentan)
E-Table 36.2 Accession Numbers of EDN1 Species
DNA
cDNA
Protein
Human Chimpanzee Mouse Rat Cow
AY434104
Y00749.1 GABD01002912 BC029547 M64711 X52740 X52942 S37093 BC102459 AF329468 X59931 S82654 X07383 AB115087 AB197698
CAA68718 JAA30188 EDL40983 EDL98206 CAA36954 CAA37117 AAB22241 AAI02460 AAG49531 CAA42555 AAB46735 CAA30296 BAD02920 BAD83370
EF127993 AF281858 BC162539
ABO33469 AF281858_1 AAI62539
Sheep Rabbit Guinea pig Pig Dog Chicken Xenopus Zebrafish
[Ala1,3,11,15] ET-1 BQ3020 IRL-1620 S6c.
Species
DNA
cDNA
Protein
Human
D11151 BAA01920
X61950.1 D90348.1 S57498.1 L06622.1 AY275462.1 AK312812.1 BC022511.1 JV044575.1
BAA01920
CM001257.1
AACZ03032416.1 AACZ03032417.1
Giant panda
ACTA01112437 ACTA01120437
Mouse Rat Cow Sheep Rabbit
Pig Dog
Chicken
AAGW02045813.1 AAGW02045814.1 AAGW02045815.1 AAEX03010018.1 AAEX03010019.1 AY438635.1 (Exons 6 and 7) AADN03004059.1
Xenopus Little brown bat Killifish Zebrafish
AADN03001977
Species
DNA
cDNA
Protein
Human
D13168.2 AY547312.1 AL139002.18 CH471093.1 CH471093.1 AJ458188.1 (Exon1) AJ458189.1 (Exon2) AJ458190.1 (Exon3) AJ458191.1 (Exon4)
M74921.1 D90402.1 S44866.1 S57283.1 L06623.1 X99250.1 AF114165.1 AY275463.1 AB209198.1 AK290699.1 BC014472.1
AAA58465.1 BAA14398.1 AAB19411.1 AAB25531.1 BAA02445.1 AAA52342.1 AAP32295.1 BAF83388.1 AAS38516.1 EAW80575.1
Donkey Crab-eating macaque Mouse
JF718901.1
EHH26227.1
JV635771.1
Chimpanzee
X14610(partial)
E-Table 36.4 Accession Numbers of EDNRB
E-Table 36.3 Accession Numbers of EDNRA
Crab-eating macaque
CH466546 CH473977.1
AFI34646.1 AFJ71111.1 GABC01004722.1 JAA06616.1 GABF01009438.1 JAA12707.1 GABD01002709.1 JAA30391.1 GABE01006931.1 JAA37808.1
AK043210.1 BC008277.1 M60786.1 X57765 BC133407 BC142309 AF416703.1 AB303551.1
BAC31493.1 AAH08277.1 AAA41114.1 CAA40917 AAI33408 AAI42310 AAL08564.1 BAF62262.1
S80652.1 AB183284
AAB36014.1 BAD83849 AAR99473.1
AF040634.1 AF472618 BC044316.1 DQ523688.1
AAC77793.1 AAM74025.1 AAH44316.1 ABF67648.1
EU391601 BC162564.1
ABY86757 AAI62564.1
Rat Guinea pig Giant panda Cow
AAKN02009242.1 ACTA01052257.1 D10994.2
Horse Zebra Goat Sheep Rabbit Pig Dog Chicken Japanese quail Little brown bat Xenopus
U20578.1
AEU10887.1 AAA62437.1
U32329.1 AK076426.1 AK082103.1 AK083415.1 AK085532.1 BC026553.1 X57764.1 S65355.1
AAB60508.1 BAC36337.1 BAC38409.1 BAC38908.1 BAC39465.1 AAH26553.1 CAA40916.1 AAB28172.1
D90456.1 BC120256.1
AF245469.1 AB209953.1 AB183285.1 AB697061.1 AB697060.1 X99295.1
BAA01762.1 BAA14422.1 AAI20257.1 AAC25983.1 AAC23486.1 AEU10892.1 BAJ77121.1 AGE10461.1 AAF60366.1 BAE48719.1 BAD83850.1 BAO52970.1 BAO52969.1 CAA67681.1
BC048223.1
AAH48223.1
AF019072.1 AF038900.1 JF718906.1 AB609187.1 JQ937242.1
AADN03000442.1 AADN03004047.1 AAPE02012009.1
AAPE02001529 AAPE02001530
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Endothelins Hirokazu Ohtaki
Abbreviation: ET, EDN Endothelin (ET), also called ET-1, was first isolated from culture supernatant of porcine endothelial cells. The hormone shows a potent vasoconstructive activity. There are three isopeptides, ET-1, -2 and -3.
Discovery Presence of a potent vasoconstructor in the supernatant of porcine endothelial cells was reported and isolated as ET-1 in 1988 [1].
Structure Structural Features ET-1 consists of 21 aa residues with free amino- and carboxyl-termini including a hydrophobic C-terminus and four cysteine residues which form two disulfide bonds (Figure 36.1). There are two isopeptides, ET-2, and ET-3, differing by the 2nd and 6th amino acids, respectively. ET-1 is the most abundant isoform.
Primary Structure Mature ET-1 is conserved (only one aa difference) in fish and the higher organisms.
Properties Mr of porcine ET-1 is 2,492 and predicted pI is 9.5. Soluble in water. ET-1 solution in water at 1 mg/ml is stable for more than a year at 220 C.
Synthesis and Release Gene and mRNA In the human, the ET-1 (EDN1), ET-2 (EDN2), and ET-3 (EDN3) genes are located on chromosome-6 (6p24.1), -1 (1p34), and -20 (20q13.2-q13.3). Human ET-1, ET-2 and ET-3 mRNA have 2109, 1258, and 2438 bp respectively. Genetic fine mapping studies of human EDN1 localized it to the telomeric region of chromosome 6p, close to the gene encoding the α-subunit of clotting factor XIII. The human EDN1 contains five exons, four introns, and 50 - and 30 -flanking regions and spans approximately 6.8 kb of DNA [2]. The ET-1 mRNA is 2.3 kb in length and directs translation of the precursor prepro-ET-1 peptide, including the sequence of pro-ET-1 (big ET) and the ET-like peptide (ETLP). The ET-1 promoter contains two functional transcription start sites including 326
TATAA and CAAT boxes, nuclear factor -1 (NF-1), GATA-2, AP-1/jun, and acute phase reaction regulatory elements (APRE).
Precursors All three ET precursors are processed by two proteases to digest the mature active forms [3]. For ET-1, the 212-residue preproendothelin, including 19 residues of signal sequence which is characteristic of a secretary signal sequence, are cleaved at the dibasic site by fulin-like endopoptidase to produce big endothelins (big ETs). The big ETs consist of 37 to 41 aa residues and are an inactive intermediate form. Mature and active ET-1 is formed by a family of membrane-bound zinc metalloproteinases, from the neprilysin superfamily, termed endothelin-converting enzyme (ECEs) [4]. Other enzymes such as non-ECE metalloproteinase, cathepsin A, and chymase contribute to the degradation.
Tissue Distribution of mRNA ET-1 is most abundantly localized in vascular endothelial cells, including umbilical vein, mesenteric artery, glomerulus, corpus cavernosum, aorta, and brain microvessels [1,2]. However, ET isopeptides are expressed in a variety of tissues and cell types and the isopeptides express a tissue-specific pattern. ET-1 mRNA expression is observed in breast epithelium, keratinocytes, endometrial stromal and glandular epithelial cells, macrophages, bone marrow mast cells, astrocytes, mesangial cells, neurons of the spinal cord and dorsal root ganglia, avascular human amnion, cardiomyocytes, and some tumor-derived cells. Few data are available for ET-2 and ET-3 in specific cell types, although a human renal adenocarcinoma cell line was shown to express ET-2. At the tissue level of rat, ET-1 expresses in vascular endothelium, lung, brain, uterus, stomach, heart, adrenal gland, and kidney [5]. ET-3 mRNA expresses in eyeball, submandibular gland, brain, kidney, jejunum, stomach, and spleen with northern blotting. Although ET-2 mRNA expresses in large and small intestine, skeletal muscle, heart, and stomach, large and small intestine expression is greatest.
Tissue and Plasma Concentrations Plasma level of ET-1 in healthy human is B2.0 pg/ml by specific radioimmunoassay (RIA). Other studies of ET-1 levels with RIA demonstrated in rat that the inner medulla of the kidney had the highest concentration (8.7 6 2.2 pg/mg of wet weight).
Y. Takei, H. Ando, & K. Tsutsui (Eds): Handbook of Hormones. DOI: http://dx.doi.org/10.1016/B978-0-12-801028-0.00036-2 © 2016 Elsevier Inc. All rights reserved.
C H A P T E R 3 6 Endothelins Biological Functions Target Cells/Tissues and Functions ET-1 causes strong and lasting vasoconstriction and raises blood pressure. Therefore, ET-1 was expected to contribute to the functioning of the cardiovascular system. Moreover, in addition to vasoconstriction, ET-1 acts on various cardiovascular cells directly or indirectly causing cell proliferation and production of diverse active substances, including extracellular matrix. ET-1 is involved in renal diseases, cancer, diabetes and insulin resistance, allograft rejection, and renal diseases, as well as cardiovascular diseases. Figure 36.1 Primary structure of mammal endothelins. Ser4 is substituted with Asn4 in mouse.
Regulation of Synthesis and Release ET-1 is transcriptionally regulated by stimuli such as shear stress, hypoxia, cytokines, lipopolysaccharide, and growth factors. These enhance ET-1 mRNA transcription and protein secretion; ET-1 shows autocrine activity. Some of them may contribute to the stability of preproET-1 mRNA.
Receptors Structure and Subtype Two G protein-coupled 7-transmembrane receptors (ETA and ETB) have been identified in humans. Both receptor types contain 7-transmembrane domain 2226 hydrophobic aa in their B400-aa sequences. The ETA exhibits subnanomolar affinities for ET-1 and ET-2 and 100-fold lower affinity for ET3 [3]. The ETB receptor has equal subnanomolar affinities for all ETs. The ETA is considered the primary vasoconstrictor and growth-promoting receptor, and the ETB inhibits cell growth and vasoconstriction in the vascular system. The ETB also functions as a “clearance receptor” which metabolites ET-1. This clearance mechanism is particularly important in the lung with metabolites being about 80% of ET-1 in the circulation.
Signal Transduction Pathway After ET-1 binding to ETA on smooth muscle cells, PLC is activated via G-coupled protein and produces IP3 and DG. IP3 induces the release of Ca21 in endoplasmic reticulum. Activation of ETA also induces the influx of extracellular Ca21 via the voltage-dependent Ca21 channel, receptor-operated Ca21 channel, and nonselective cation channel. The Ca21 influx increases [Ca21]i level and finally induces the phosphorylation of the myosin light chain and leads to contraction of the smooth muscle cells.
Agonist No synthetic agonist is shown for ETA. Agonists for ETB are {Ala1,3,11,15} ET-1, BQ3020, IRL-1620, and S6c.
Antagonist Peptide and non-peptide ET receptor selective or nonselective antagonists have been developed by pharmaceutical companies. Ro47-0203 (Bosentan) is a non-selective ET receptor antagonist used in the treatment of pulmonary artery hypertension (PAH). Representative selective ETA receptor antagonists are BQ-123, BMS182874, LU135252 (darusentan) and PD156707. Representative selective ETB receptor antagonists are BQ-788 and RES701-1. Representative non-selective ETA/ETB receptor antagonists are TAK-044 and Ro47-0203 (bosentan).
Phenotype in Gene-Modified Animals A series of gene-deficient mice of ETs and the receptors are reported from the Yanagisawa group. The ligand or receptor gene-deficient mice showed several developmental abnormalities. Homozygous ET-1 knockout mice have craniofacial and cardiac malformations that lead to neonatal death [6]. ET-2 knockout mice exhibit growth retardation, hypothermia, hypoxemic hypoxia, hypercapnia, emphysema, and premature death [7]. ET-3 knockout mice exhibit aganglionic megacolon with white spotting of the hair coat due to impaired expansion and differentiation of epidermal melanoblasts. Mutants die around weaning with impacted colons [8]. Homozygous inactivation of ETA causes perinatal lethality and craniofacial deformities including middle ear defects. The knockout mice also show severe cardiac outflow and great vessel anomalies, respiratory and PNS defects, thymus and tongue hypoplasia, and absent salivary glands [9]. ETB knockout mice show pigmentation limited to small patches on the head and rump, exhibit abnormal neural epithelium of the inner ear, and die from megacolon resulting from impaired neuronal migration and aganglia [10].
Pathophysiological Implications Clinical Implications Plasma ET1 level was measured in diverse abnormal states of humans with RIA and was reported to increase in abnormal states such as pulmonary hypertension, acute myocardial infarction, essential hypertension, subarachnoid hemorrhage, and diabetes mellitus. The plasma level of ET-1 increases with the severity of congestive heart failure (CHF) and, in particular, increases in patients nominated as Class IV of the New York Heart Association (NYHA) functional classification. The plasma ET-1 and big-ET-1 levels are inversely correlated with the left ventricle ejection fraction (LVEF) and are independent predictors of survival in patients with severe CHF.
Use for Diagnosis and Treatment ET-1 concentration is not measured to diagnosis in clinical settings. A non-selective ETA/ETB antagonist (bosentan; Tracleers) is used for treatment of pulmonary hypertension. A selective ETA antagonist (ambrisentan; Volibriss) has also been used for the disease since 2010. References 1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411415. 2. Rubanyi GM, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev. 1994;46:325415. 3. Kedzierski RM, Yanagisawa M. Endothelin system: the doubleedged sword in health and disease. Annu Rev Pharmacol Toxicol. 2001;41:851876.
327
P A R T I Peptides and Proteins in Vertebrates 4. Barton M, Yanagisawa M. Endothelin: 20 years from discovery to therapy. Can J Physiol Pharmacol. 2008;86:485498. 5. Sakurai T, Masaki T. Endothelin receptors—an overview. Tanpakushitsu Kakusan Koso. 1991;36:23812388. 6. Kurihara Y, Kurihara H, Suzuki H, et al. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature. 1994;368:703710. 7. Chang I, Bramall AN, Baynash AG, et al. Endothelin-2 deficiency causes growth retardation, hypothermia, and emphysema in mice. J Clin Invest. 2013;123:26432653.
8. Baynash AG, Hosoda K, Giaid A, et al. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell. 1994;79:12771285. 9. Clouthier DE, Hosoda K, Richardson JA, et al. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development. 1998;125:813824. 10. Hosoda K, Hammer RE, Richardson JA, et al. Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell. 1994;79:12671276.
Supplemental Information
E-Figure 36.1 Human endothelin-1 (EDN1) gene located on chromosome 6 (6p24.1).
328
C H A P T E R 3 6 Endothelins
E-Figure 36.2 The alignment of amino acid sequence of the EDN1 in vertebrates. Conserved amino acid residues are indicated in green, and Cys residues are indicated in yellow. Mature endothelin-1 is conserved in (only one aa difference) in fish and the higher organisms. Accession numbers: human, NP_001161791; chimpanzee, XP_518241; monkey, XP_001089874; dog, NP_001002956; cow, NP_851353; mouse, NP_034234; rat, NP_036680; chicken, XP_418943; frog, XP_002932710; zebrafish, NP_571594.
E-Figure 36.3 Human endothelin-1 receptor type A (EDNRA) gene located on chromosome 4 (4q31.22).
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P A R T I Peptides and Proteins in Vertebrates
E-Figure 36.4 Human endothelin-1 receptor type B (EDNRB) gene located on chromosome 13 (13q22).
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C H A P T E R 3 6 Endothelins
E-Figure 36.5 The alignment of amino acid sequence of the EDNRA in vertebrates. Conserved amino acid residues are indicated in green, and Cys residues are indicated in yellow. Accession numbers: human, NP_001948; chimpanzee, XP_001149597; monkey, NP_001248421, XP_001098827; dog, NP_001026802 XP_539756; cow, NP_776733; mouse, NP_034462, XP_134499; rat, NP_036682, XP_001054233; chicken, NP_989450; frog, NP_001072275; zebrafish, NP_001092915, XP_687609.
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P A R T I Peptides and Proteins in Vertebrates
E-Figure 36.6 The alignment of amino acid sequence of the EDNRB in vertebrates. Conserved amino acid residues are indicated in green, and Cys residues are indicated in yellow. Accession numbers: human, NP_001188326; chimpanzee, XP_509693; monkey, XP_001089047; dog, NP_001010943; cow, NP_776734; mouse, NP_001129533; rat, NP_059029, XP_344452; chicken; NP_001001127, XP_417001; frog, NP_001072476; zebrafish, NP_571272.
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C H A P T E R 3 6 Endothelins E-Table 36.1 Lists of Agonists and Antagonists for ETRs Selective ETA antagonist peptide
BQ-123 BQ-610 FR139317 TTA 788 nonpeptide ABT-627 (arasentan) BMS182874 LU135252 (darusentan) PD156707 PD176856 Ro61-612 (tezosentan) Ro61-1790 S-0139 EMD122946 SB247083 TBC11251 (sitaxsentan) ZD1611 T-0201 J-104121 J-104131 YM598 N/A
agonist
Selective ETB
Nonselective ETA/ETB
BQ-788 IRL2500 RES701-1
PD142893 TAK044
A-192621
A-182086
K-8794
L-754142 Ro46-2005 Ro47-0203 (bosentan) SB209670 SB217242 (enrasentan)
E-Table 36.2 Accession Numbers of EDN1 Species
DNA
cDNA
Protein
Human Chimpanzee Mouse Rat Cow
AY434104
Y00749.1 GABD01002912 BC029547 M64711 X52740 X52942 S37093 BC102459 AF329468 X59931 S82654 X07383 AB115087 AB197698
CAA68718 JAA30188 EDL40983 EDL98206 CAA36954 CAA37117 AAB22241 AAI02460 AAG49531 CAA42555 AAB46735 CAA30296 BAD02920 BAD83370
EF127993 AF281858 BC162539
ABO33469 AF281858_1 AAI62539
Sheep Rabbit Guinea pig Pig Dog Chicken Xenopus Zebrafish
[Ala1,3,11,15] ET-1 BQ3020 IRL-1620 S6c.
Species
DNA
cDNA
Protein
Human
D11151 BAA01920
X61950.1 D90348.1 S57498.1 L06622.1 AY275462.1 AK312812.1 BC022511.1 JV044575.1
BAA01920
CM001257.1
AACZ03032416.1 AACZ03032417.1
Giant panda
ACTA01112437 ACTA01120437
Mouse Rat Cow Sheep Rabbit
Pig Dog
Chicken
AAGW02045813.1 AAGW02045814.1 AAGW02045815.1 AAEX03010018.1 AAEX03010019.1 AY438635.1 (Exons 6 and 7) AADN03004059.1
Xenopus Little brown bat Killifish Zebrafish
AADN03001977
Species
DNA
cDNA
Protein
Human
D13168.2 AY547312.1 AL139002.18 CH471093.1 CH471093.1 AJ458188.1 (Exon1) AJ458189.1 (Exon2) AJ458190.1 (Exon3) AJ458191.1 (Exon4)
M74921.1 D90402.1 S44866.1 S57283.1 L06623.1 X99250.1 AF114165.1 AY275463.1 AB209198.1 AK290699.1 BC014472.1
AAA58465.1 BAA14398.1 AAB19411.1 AAB25531.1 BAA02445.1 AAA52342.1 AAP32295.1 BAF83388.1 AAS38516.1 EAW80575.1
Donkey Crab-eating macaque Mouse
JF718901.1
EHH26227.1
JV635771.1
Chimpanzee
X14610(partial)
E-Table 36.4 Accession Numbers of EDNRB
E-Table 36.3 Accession Numbers of EDNRA
Crab-eating macaque
CH466546 CH473977.1
AFI34646.1 AFJ71111.1 GABC01004722.1 JAA06616.1 GABF01009438.1 JAA12707.1 GABD01002709.1 JAA30391.1 GABE01006931.1 JAA37808.1
AK043210.1 BC008277.1 M60786.1 X57765 BC133407 BC142309 AF416703.1 AB303551.1
BAC31493.1 AAH08277.1 AAA41114.1 CAA40917 AAI33408 AAI42310 AAL08564.1 BAF62262.1
S80652.1 AB183284
AAB36014.1 BAD83849 AAR99473.1
AF040634.1 AF472618 BC044316.1 DQ523688.1
AAC77793.1 AAM74025.1 AAH44316.1 ABF67648.1
EU391601 BC162564.1
ABY86757 AAI62564.1
Rat Guinea pig Giant panda Cow
AAKN02009242.1 ACTA01052257.1 D10994.2
Horse Zebra Goat Sheep Rabbit Pig Dog Chicken Japanese quail Little brown bat Xenopus
U20578.1
AEU10887.1 AAA62437.1
U32329.1 AK076426.1 AK082103.1 AK083415.1 AK085532.1 BC026553.1 X57764.1 S65355.1
AAB60508.1 BAC36337.1 BAC38409.1 BAC38908.1 BAC39465.1 AAH26553.1 CAA40916.1 AAB28172.1
D90456.1 BC120256.1
AF245469.1 AB209953.1 AB183285.1 AB697061.1 AB697060.1 X99295.1
BAA01762.1 BAA14422.1 AAI20257.1 AAC25983.1 AAC23486.1 AEU10892.1 BAJ77121.1 AGE10461.1 AAF60366.1 BAE48719.1 BAD83850.1 BAO52970.1 BAO52969.1 CAA67681.1
BC048223.1
AAH48223.1
AF019072.1 AF038900.1 JF718906.1 AB609187.1 JQ937242.1
AADN03000442.1 AADN03004047.1 AAPE02012009.1
AAPE02001529 AAPE02001530
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