Structural organization and chromosomal assignment of the gene encoding endothelin

Endothelin is a 21-amino acid vasoconstrictor synthesized and secreted by vascular endothelial cells. The human peptide is derived from a 212-amino acid precursor, preproendothelin. A nearly full length clone containing DNA complementary to human preproendothelin mRNA was isolated, and its nucleotide sequence was determined. Using this cDNA as a probe, the genomic organization of the human endothelin gene was determined and the promoter region delineated. The gene contains five exons and four intervening sequences. Nucleotide sequences encoding endothelin are contained within the second exon, and the third exon specifies a portion of preproendothelin that is homologous to endothelin. The second and third exons may represent descendants of a common progenitor exon. The 3'-untranslated portion of the gene contains a 250-base pair region that is highly conserved between human and porcine genomes and may have an important role in endothelin mRNA stability. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, the endothelin gene was assigned to human chromosome 6.     

Solid phase peptide synthesis of human endothelin precursor peptides using two-step hard acid deprotection/cleavage methods.

Syntheses are described for the putative human and porcine biosynthetic precursors (hET-38 and pET-39) of endothelin, with the sequence previously deduced from human- and porcine-cDNA coding for preproendothelin. The Boc based solid phase synthetic method was applied, followed by weak hard acid, trimethylsilyl bromide, cleavage. The peptide removal from the resin was optimally accomplished with hydrogen fluoride. Disulfide bridges were formed by air-oxidation, and the linkage modes determined by enzymic (Endoproteinase Asp-N) digestion and HPLC. Five additional C-terminally elongated endothelin homologs were also synthesized. For alternative synthesis of pET-39, the use of trimethylsilyl trifluoromethanesulfonate for the removal of peptide from the resin generated a major side product, which was characterized. hET-38 was found to be less effective in vitro, when compared to endothelin. The vasoconstrictor activity in vitro of other related peptides was comparable to that of hET-38.     

Cloning and functional expression of human cDNA for the ETB endothelin receptor.

We report the cloning of human cDNA encoding an ETB (non-isopeptide-selective) subtype of the endothelin receptor. The predicted amino acid sequence of the human ETB endothelin receptor was 87.8% and 62.9% identical with the previously cloned rat ETB and ETA receptors, respectively. COS cells transiently transfected with the cloned cDNA expressed specific, high-affinity binding sites for endothelin isopeptides and responded to the peptides with a transient increase of [Ca2+]i; endothelin-1 and endothelin-3 exhibited approximately equal potencies both in displacing 125I-labeled endothelin-1 binding and in eliciting [Ca2+]i transients. The ETB receptor mRNAs were expressed in various human tissues and also in the intact porcine aortic intimal cells ex vivo.     

Chromosomal assignments of the human endothelin family genes: the endothelin-1 gene (EDN1) to 6p23-p24, the endothelin-2 gene (EDN2) to 1p34, and the endothelin-3 gene (EDN3) to 20q13.2-q13.3.

Chromosomal assignments of the genes for the human endothelin family, the endothelin-1 gene (EDN1), the endothelin-2 gene (EDN2), and the endothelin-3 gene (EDN3), were accomplished by in situ hybridization to human metaphase chromosomes using a 3H-labeled human preproendothelin cDNA probe for each endothelin. The chromosomal assignment of the EDN2 was also performed by Southern blot analysis of somatic cell hybrid DNAs. EDN1, EDN2, and EDN3 were mapped to 6p23-p24, 1p34, and 20q13.2-q13.3, respectively.     

Cloning and chromosomal localization of a human endothelin ETA receptor.

A cDNA clone encoding a human endothelin receptor was isolated from a placenta cDNA library. The deduced amino acid sequence of the clone is 94% identical to the bovine endothelin ETA receptor and represents the human homologue. The human endothelin ETA receptor gene was localized to chromosome 4 by analysis of its segregation pattern in rodent-human hybrids.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Chromatographic characterization of immunoreactive endothelin in rat lung

Using a specific and sensitive radioimmunoassay for endothelin, combined with gel filtration and reverse phase high performance liquid chromatography, the molecular form of immunoreactive endothelin in the rat lung was investigated. On reverse phase high performance chromatography, the major immunoreactive endothelin in the rat lung emerged at a position identical to that of authentic endothelin-1. After oxidation of the immunoreactive endothelin by H2O2, the immunoreactivity migrated to a position identical to that of Met-sulfoxide endothelin-1. These data indicate that the major immunoreactive endothelin in the rat lung is not endothelin-3 (putative rat/human endothelin), but is identical or very similar to endothelin-1 (porcine/human endothelin).     

Cloning and characterization of cDNA encoding human A-type endothelin receptor.

A cDNA coding for the human A-type endothelin receptor (ETA) was cloned from a human placenta cDNA library. The cDNA contained the entire coding sequence for the 427 amino acid protein with a relative Mr of 48,722. The deduced amino acid sequence of the human ETA was, respectively, 94% and 93% homologous with the sequence of bovine ETA and rat ETA, but was only 64% homologous with that of the human ETB receptor. Upon expression in COS-1 cells, the human ETA receptor showed binding activity to ETA, with the highest selectivity to ET-1. Northern blot analysis showed that the mRNA of human placenta ETA consists of one species 5 kilo-nucleotides in length, and the same analysis for the uterus, testis, heart and adrenal gland of Cynomolgus monkey showed that the cognate mRNAs are widely distributed.     

Cloning and sequence analysis of a cDNA encoding human non-selective type of endothelin receptor.

A cDNA encoding non-selective type (ETB) of endothelin receptor was isolated from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 442 amino acid residues with a relative Mr of 49,643. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and bovine lung ET-1-specific (ETA) receptor, respectively, and contained a relatively long and proline-rich extracellular N-terminal region in addition to a significant similarity with the G protein-coupled receptor super-family with seven transmembrane segments.     

Molecular cloning of a non-isopeptide-selective human endothelin receptor.

We isolated several complementary DNA (cDNA) clones encoding a non-isopeptide-selective human endothelin receptor (ETBR) from a human placenta cDNA library. The clones, different in the length of their 3'-untranslated regions, encoded the same 442-amino acid protein with a transmembrane topology similar to that of other G protein-coupled receptors. The rank order of the binding of ET isopeptides (ET-1, ET-2 and ET-3) to the receptor expressed in COS-7 cells was ET-1 = ET-2 = ET-3. Northern blot analysis identified three mRNA species, 4.3 kb, 2.7 kb and 1.7 kb in size, probably generated by their use of alternative polyadenylation sites. These mRNAs were expressed in a wide variety of human tissues, at the highest level in the brain and at a significant level in cultured endothelial cells.     

Synthesis of the vasoconstrictor peptide endothelin in kidney cells

The expression plasmid containing human prepro-endothelin cDNA was constructed and introduced into COS-7 cells. Mature endthelin, consisting of 21 amino acid residues, was secreted into the culture medium of the transfected cells and was also synthesized by non-transfected COS-7 cells. Normal kidney cells derived from other species also synthesized and secreted endothelin. Partial characterization of endothelins produced by kidney cells suggested that existence of new types of endothelin. This is the first report of the vasoconstrictor peptide endothelin being synthesized in kidney cells.     

Synthesis and disulfide structure determination of porcine endothelin: an endothelium-derived vasoconstricting peptide

All disulfide analogs (types A, B and C) of porcine or human endothelin, a 21-amino acid peptide having two intramolecular disulfide bonds, were synthesized and their retention times on HPLC were compared with that of natural endothelin. One of the analogs (type A) having disulfide bonds between positions 1 and 15 and between 3 and 11 was found to be identical with natural endothelin. Random oxidation of fully reduced endothelin formed a mixture of type A and B in a ratio of 3:1, with almost none of type C, which has disulfide bonds between positions 1 and 3 and between 11 and 15. Type A endothelin was also synthesized by the segment condensation procedure in solution applying our maximum protection strategy. This product was found to have full vasoconstricting activity in rat pulmonary artery ring preparations; the potency was as high as that of the natural product.     

Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain

The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immunocytochemistry. Cryostat sections from 22 cases of neurologically normal adult human brain, collected 3-7 h post-mortem were hybridized with 35S-labelled complementary (c)RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae III-VI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scattered throughout the para- and periventricular, supraoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunoreactive cells throughout the brain, were noted. Immunoreactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochemistry on the same section revealed the presence of endothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cortex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreactivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. Endothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogenous neuronal populations further emphasises the potential role of endothelin as a neuropeptide, probably having diverse actions in the nervous system of man.     

cDNA cloning and chromosomal assignment of the endothelin 2 gene: vasoactive intestinal contractor peptide is rat endothelin 2.

Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.     

Potent vasoactive properties of endothelin 1 in human skin.

The effect of the endothelial cell-derived peptide endothelin 1 was investigated in human skin. Intradermal injection of endothelin 1 (1-100 pmol) caused a dose-dependent area of pallor that was associated with a significant reduction in basal skin blood flow, measured by laser-Doppler blood flowmeter (with 1 pmol endothelin, P = 0.012, analysis of variance). The coadministration of endothelin 1 (1-100 pmol) with the neuropeptide vasodilator calcitonin gene-related peptide (CGRP) inhibited the vasodilator response to CGRP (10 pmol) by up to 82.7 +/- 9.2% (with 100 pmol endothelin, P less than 0.001). The response of the prostanoid vasodilator prostaglandin E2 (10 pmol) was inhibited by endothelin in a similar manner. In addition to the vasoconstrictor effects, endothelin 1 produced a dose-dependent flare that surrounded the area of pallor, and this was associated with a significant increase in blood flow (P less than 0.05) within the flare area. The H1 antagonist terfenadine (120 mg po) significantly reduced the flare area associated with endothelin 1: flare 5 min after intradermal endothelin (10 pmol, placebo treated), 668 +/- 405 mm2; terfenadine treated, 201 +/- 257 mm2 (P less than 0.05). The flare was also significantly attenuated when endothelin (10 pmol) was injected into local anesthetic-treated skin. Thus intradermal injection of endothelin in humans causes long-lasting vasoconstriction at the site of injection and a surrounding flare. Results suggest that the flare component is partially histamine dependent and the result of an axon reflex. This study demonstrates the potent activity of endothelin in human skin. It is possible that endothelin could be relevant to the local response of skin to injury.     

Conversion of porcine big endothelin to endothelin by an extract from the porcine aortic endothelial cells

Conversion of porcine big endothelin (big ET) to endothelin (ET) by an extract from cultured porcine aortic endothelial cells was investigated using a radioimmunoassay (RIA) specific for ET and reverse-phase high performance liquid chromatography (RP-HPLC). When big ET was incubated with the extract at an acid pH in the presence of E-64, a cysteine protease inhibitor, the amount of immunoreactive-ET (IR-ET) in the incubation mixture was greatly increased and the optimum pH for this increased reaction was 4.0. The extract-induced increase in IR-ET was completely inhibited by pepstatin-A. These immunoreactive alterations correlated with those in the vasoconstrictor activity. When the incubation mixture of big ET with the cell extract was applied to the RP-HPLC, the IR-ET eluted at the same retention time as seen with synthetic porcine ET. We suggest that a pepstatin-sensitive aspartic protease is involved in the conversion of big ET to ET in vascular endothelial cells.     

Formation of endothelin by cultured airway epithelial cells

Immunoreactivity to endothelin was detected in conditioned culture medium from both canine and porcine tracheal epithelial cells. Gel permeation chromatography and fast protein liquid chromatography were used to confirm the identity of the endothelin. The two peaks demonstrated on fast protein liquid chromatography co-eluted with endothelin 1 and endothelin 3 respectively.     

Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain

Summary The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immunocytochemistry. Cryostat sections from 22 cases of neurologically normal adult human brain, collected 3–7 h post-mortem were hybridized with³⁵S-labelled complementary (c)RNA probes prepared from the 3 non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae III–VI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scattered throughout the para- and periventricular; supraoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunoreactive cells throughout the brain, were noted. Immunoreactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochemistry on the same section revealed the presence of endothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cortex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreactivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. Endothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogenous neuronal populations further emphasises the potential role of endothelin as a neuropeptide, probably having diverse actions in the nervous system of man.     

Identification of [I]endothelin binding sites in human coronary tissue

In vitro receptor autoradiography has been used to study the distribution of [¹²⁵I]endothelin binding sites in human coronary tissue from patients undergoing cardiac transplantation. Dense binding of [¹²⁵I]endothelin was associated with the smooth muscle of epicardial coronary arteries as well as to perivascular regions of these vessels. Binding was also associated with the ventricular myocardium. There was an increased binding of [¹²⁵I]endothelin to atheromatous tissue, both coronary arteries and vein graft.

The [¹²⁵I]endothelin binding sites identified using in vitro autoradiography are likely to be functionally relevant since endothelin causes a concentration-dependent contraction of segments of human epicardial coronary arteries in vitro and also has positive inotropic activity on isolated human cardiomyocytes.

The presence of specific binding sites for [¹²⁵I]endothelin on coronary tissue and the increased binding in atheromatous tissue suggest that endothelin is a peptide which may play a role in the maintenance of vascular tone and/or the pathogenesis of ischaemic heart disease.