A prolactin release inhibiting activity isolated from neurointermediate lobe extracts is an endothelin-like peptide.

A prolactin release-inhibiting factor has been reported in extracts of rat and bovine neurointermediate lobes of the pituitary gland. This material when isolated by Sephadex G-25 chromatography or by C-8 high pressure liquid chromatography coelutes with synthetic forms of all members of the mammalian endothelin family of peptides and with their immediate precursors, big-ET-1 and big-ET-3. The endogenous material crossreacts in an endothelin radioimmunoassay. Immunohistochemical analysis of endothelin-like immunoreactivity in the neurointermediate lobe revealed a predominantly intermediate lobe localization. Individual immunopositive cells were visualized in the intermediate lobe. Preabsorption of chromatographically isolated prolactin release-inhibiting activity with an endothelin antiserum which recognizes all members of the ET family of peptides abolishes its in vitro bioactivity. These results suggest that a prolactin release-inhibiting activity present in the neurointermediate lobe is endothelin or an endothelin-like peptide.     

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.     

EDNRB/EDN3 and Hirschsprung disease type II.

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1-4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah-Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type-B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest-derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest-derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB-mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3-induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post-coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3--a receptor-mediated pathway with pleiotropic effects.     

EDNRB/EDN3 and Hirschsprung Disease Type II

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1–4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah‐Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type‐B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest‐derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest‐derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB‐mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3‐induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post‐coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3 – a receptor‐mediated pathway with pleiotropic effects.     

Neural crest cell plasticity and its limits

The neural crest (NC) yields pluripotent cells endowed with migratory properties. They give rise to neurons, glia, melanocytes and endocrine cells, and to diverse 'mesenchymal' derivatives. Experiments in avian embryos have revealed that the differentiation of the NC 'neural' precursors is strongly influenced by environmental cues. The reversibility of differentiated cells (such as melanocytes or glia) to a pluripotent precursor state can even be induced in vitro by a cytokine, endothelin 3. The fate of 'mesenchymal' NC precursors is strongly restricted by Hox gene expression. In this context, however, facial skeleton morphogenesis is under the control of a multistep crosstalk between the epithelia (endoderm and ectoderm) and NC cells.     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

A comparative study of endothelin and sarafotoxin action in vascular and non-vascular smooth muscle

The effects of endothelin and sarafotoxin on smooth muscle tone have been examined in the rat aorta and anococcygeus muscle and their actions compared to those of norepinephrine. The contractions elicited by endothelin and sarafotoxin (10 nM), or norepinephrine (1 μM) were approximately equieffective in terms of tension development and correspond to EC₅₀ values and these concentrations were thus used throughout the study. In calcium-free Krebs the three agonists generated approximately similar levels of tone in the aorta and the anococcygeus corresponding to 20 and 5% of the maximum response, respectively. Nifedipine, 10 μM, significantly inhibited responses to endothelin and norepinephrine in the aorta but only norepinephrine in anococcygeus; the responses to sarafotoxin were however not significantly affected in either tissue. A combination of 10 μM ryanodine and nifedipine caused near complete inhibition of the response to endothelin in the aorta and also significantly reduced the response to both endothelin and norepinephrine in the anococcygeus. The lipoxgenase inhibitor, nordihydroguaiaretic acid, inhibited the response to endothelin in the aorta and endothelin and norepinephrine in the anococcygeus muscle. The cyclooxygenase inhibitor, indomethacin, however, had no effect on the responses to any of the three agonists in either the aorta or anococcygeus. At concentrations greater than 30 nM both endothelin and sarafotoxin induced myogenic activity in normally quiescent anococcygeus muscle. As determined by the loss of myogenic activity the tissues recovered more rapidly from sarafotoxin than endothelin with complete recovery apparent after 2.62 ± 0.85 and 5.22 ± 0.06 h respectively. Omitting Ca²⁺ from the Krebs solution reduced recovery times to 1.62 ± 0.2 and 2.4 ± 0.51 h respectively.

Overall the results suggest that endothelin and sarafotoxin activate different cell signaling systems to differing extents in rat aorta versus anococcygeus suggesting that the membrane receptors mediating the responses to endothelin and sarafotoxin are not necessarily identical.     

Induction of melanogenesis by tetradecanoylphorbol-13 acetate and endothelin 3 in embryonic avian peripheral nerve cultures

In this study, we have analyzed the melanogenic potential of Schwann cells using in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in Schwann cells, two factors, 12-O-tetradecanoylphorbol-13 acetate (TPA) and endothelin 3, trigger a differentiation pathway toward melanocytes, and that Steel factor has no effect on these cells unless treated simultaneously with TPA. In these cultures, TPA induces the expression of c-kit, whereas Steel factor enhances the development of melanocytes. In the assay system we employed, neither neuronal nor catecholaminergic phenotypes were obtained, regardless of various combinations of related factors added to the culture medium. These data support our previous observations indicating the existence of bipotent progenitors that are capable of differentiating into Schwann cells or into melanocytes, and the regulatory role of endothelin 3 on those precursors, as revealed by the clonal culture of neural crest cells.     

Induction of Melanogenesis by Tetradecanoylphorbol‐13 Acetate and Endothelin 3 in Embryonic Avian Peripheral Nerve Cultures

In this study, we have analyzed the melanogenic potential of Schwann cells using in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in Schwann cells, two factors, 12‐O‐tetradecanoylphorbol‐13 acetate (TPA) and endothelin 3, trigger a differentiation pathway toward melanocytes, and that Steel factor has no effect on these cells unless treated simultaneously with TPA. In these cultures, TPA induces the expression of c‐kit, whereas Steel factor enhances the development of melanocytes. In the assay system we employed, neither neuronal nor catecholaminergic phenotypes were obtained, regardless of various combinations of related factors added to the culture medium. These data support our previous observations indicating the existence of bipotent progenitors that are capable of differentiating into Schwann cells or into melanocytes, and the regulatory role of endothelin 3 on those precursors, as revealed by the clonal culture of neural crest cells.     

The endothelin system in human and monkey ovaries: in situ gene expression of the different components

The endothelin system is composed of three endothelin isoforms (ET-1, ET-2, and ET-3), the endothelin receptors ETA and ETB, and the endothelin-converting enzyme (ECE). Besides having a major vasoactive role, endothelins have roles in different cell types at a local level. We investigated the presence of the different components of the endothelin system in primate ovaries. Human ovaries and gonadotropin-stimulated monkey ovaries were studied using immunohistochemistry for endothelin, and in situ hybridization with probes for ET-1, ET-2, ET-3, ETA and ETB receptors, and ECE. ET-1 and ETA receptors were detected in endothelial cells and vascular smooth muscle cells, respectively, in stromal vessels adjacent to follicles and corpora lutea. ETB receptors and ET-1 were found in the endothelial cells of capillaries of corpora lutea. ECE was present in internal theca cells of secondary, de Graaf, atretic follicles, and in luteinized granulosa cells of the corpora lutea. The endothelin system components are present in or around the follicles of human and monkey ovaries. Although the components are not expressed in the same cell types, they are synthesized, mainly in follicles, by cells that are in close proximity. Thus, the endothelin system could act in a paracrine manner. ECE expression in steroid-producing cells changes its compartmentalization during follicle maturation.     

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.     

Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation

The functional and structural characterization of G-protein-coupled receptors (GPCRs) still suffers from tremendous difficulties during sample preparation. Cell-free expression has recently emerged as a promising alternative approach for the synthesis of polytopic integral membrane proteins and, in particular, for the production of G-protein-coupled receptors. We have now analyzed the quality and functional folding of cell-free produced human endothelin type B receptor samples as an example of the rhodopsin-type family of G-protein-coupled receptors in correlation with different cell-free expression modes. Human endothelin B receptor was cell-free produced as a precipitate and subsequently solubilized in detergent, or was directly synthesized in micelles of various supplied mild detergents. Purified cell-free-produced human endothelin B receptor samples were evaluated by single-particle analysis and by ligand-binding assays. The soluble human endothelin B receptor produced is predominantly present as dimeric complexes without detectable aggregation, and the quality of the sample is very similar to that of the related rhodopsin isolated from natural sources. The binding of human endothelin B receptor to its natural peptide ligand endothelin-1 is demonstrated by coelution, pull-down assays, and surface plasmon resonance assays. Systematic functional analysis of truncated human endothelin B receptor derivatives confined two key receptor functions to the membrane-localized part of human endothelin B receptor. A 39 amino acid fragment spanning residues 93-131 and including the proposed transmembrane segment 1 was identified as a central area involved in endothelin-1 binding as well as in human endothelin B receptor homo-oligomer formation. Our approach represents an efficient expression technique for G-protein-coupled receptors such as human endothelin B receptor, and might provide a valuable tool for fast structural and functional characterizations.     

Radioimmunoassay for endothelin and immunoreactive endothelin in culture medium of bovine endothelial cells

Using a synthetic 21-residue endothelin as antigen, we have produced an antiserum for endothelin and developed a specific and sensitive radioimmunoassay (RIA) for endothelin. The minimum detection limit of the RIA was 1 pg/tube. Immunoreactive (ir-) endothelin was extracted from the culture medium by Bondelute C8 column. The ir-endothelin in the culture medium of endothelial cells (EC) from bovine pulmonary artery and carotid artery was 1.48 ng/ml and 3.31 ng/ml, respectively. Reverse-phase high performance liquid chromatography coupled with the RIA revealed that ir-endothelin in the culture medium comprised one major component corresponding to synthetic endothelin. In addition, the cultured EC of bovine pulmonary artery were specifically stained by immunohistochemical technique. These results suggest that endothelin could be produced in the EC of the pulmonary and carotid arteries besides the aorta. The RIA presented in this study could be an useful tool to investigate the pathophysiologic significance of endothelin.     

Receptor subtype mediating the action of circulating endothelin on glucose metabolism and hemodynamics in perfused rat liver.

The subtype of endothelin receptor that mediates metabolic and hemodynamic effects of circulating endothelin was explored using perfused rat liver. Infusion of endothelin (ET)-1 or ET-3 into the portal vein at a concentration of 0.3 nM increased glucose and lactate output and decreased perfusion flow, although ET-3 was less effective than ET-1. The metabolic effects of ET-1 were observed even under costant-flow perfusion. Infusion of either sarafotoxin S6b or S6c, an ET(A)- or ET(B)-receptor agonist, mimicked the actions of ET-1 to an equal extent. The flow reduction and glucose production induced by ET-1 were partly attenuated by the ET(A)-receptor antagonist BQ485. By contrast, ET(B)-receptor antagonist BQ788 enhanced glucose production caused by ET-1 and ET-3 without affecting the hemodynamic change. The effects of ET-1 and ET-3 were almost totally inhibited by the combination of BQ485 and BQ788. These results suggest that both ET(A) and ET(B) receptors are involved in the metabolic and hemodynamic effects of circulating endothelin in rat liver, while the ET(A)-receptor-mediated action appears to be dominant.     

Specific cleavage of DNA at CG sites by Co(III) and Ni(II) desferal complexes.

Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse-phase HPLC and an automated gas-phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC. Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N-terminal sequences of the degradation products revealed that the phosphoramidon-sensitive fragments were generated by cleavage at the Ser5-Leu6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon-insensitive fragment was produced by cleavage at Leu17-Asp18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon-insensitive metallo-endopeptidase recently isolated from rat kidney membranes by us [(1992) Eur. J. Biochem. 204, 547-552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of Km = 71.5 microM and kcat = 1.49 s-1, giving a ratio of kcat/Km = 2.08 x 10(4) s-1.M-1. The Km value was much higher and the kcat/Km value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.     

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.     

碱性成纤维细胞生长因子增加大鼠血管一氧化氮及内皮素的生成

研究碱性成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ｂＦＧＦ）对自发性高血压大鼠（ＳＨＲ）和ＷＫＹ大鼠血管一氧化氮（ＮＯ）及内皮素生成的影响。取ＳＨＲ和ＷＫＹ大鼠主动脉制成血管薄片,以１０、１００ｎｇ／ｍｌ ｂＦＧＦ（终浓度）分别孵育６ｈ,测定血管组织中一氧化氮合酶（ＮＯＳ）活性及孵育液中亚哨酸盐（ＮＯ２）和内皮素含量。结果显示：ＳＨＲ主动脉组织ＮＯＳ活性较Ｗ