Endothelins (EDN1, EDN2, EDN3) and their receptors (EDNRA, EDNRB, EDNRB2) in chickens: Functional analysis and tissue distribution.

Endothelins (EDNs) and their receptors (EDNRs) are reported to be involved in the regulation of many physiological/pathological processes, such as cardiovascular development and functions, pulmonary hypertension, neural crest cell proliferation, differentiation and migration, pigmentation, and plumage in chickens. However, the functionality, signaling, and tissue expression of avian EDN-EDNRs have not been fully characterized, thus impeding our comprehensive understanding of their roles in this model vertebrate species. Here, we reported the cDNAs of three EDN genes (EDN1, EDN2, EDN3) and examined the functionality and expression of the three EDNs and their receptors (EDNRA, EDNRB and EDNRB2) in chickens. The results showed that: 1) chicken (c-) EDN1, EDN2, and EDN3 cDNAs were predicted to encode bioactive EDN peptides of 21 amino acids, which show remarkable degree of amino acid sequence identities (91-95%) to their respective mammalian orthologs; 2) chicken (c-) EDNRA expressed in HEK293 cells could be preferentially activated by chicken EDN1 and EDN2, monitored by the three cell-based luciferase reporter assays, indicating that cEDNRA is a functional receptor common for both cEDN1 and cEDN2. In contrast, both cEDNRB and cEDNRB2 could be activated by all three EDN peptides with similar potencies, indicating that both receptors can function as common receptors for the three EDNs and share functional similarity. Moreover, activation of three EDNRs could stimulate intracellular calcium, MAPK/ERK, and cAMP/PKA signaling pathways. 3) qPCR assay revealed that cEDNs and cEDNRs are widely, but differentially, expressed in adult chicken tissues. Taken together, our data establishes a clear molecular basis to uncover the physiological/pathological roles of EDN-EDNR system in birds and helps to reveal the conserved actions of EDN-EDNR signaling across vertebrates.

Expression and purification of an engineered human endothelin receptor B in a monomeric form.

In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.

Endothelins specifically recognize lysophosphatidylcholine micelles.

Lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low-density lipoprotein (ox-LDL), is implicated in numerous inflammatory diseases, including atherosclerosis. Here, to clarify the relationship between bioactive endothelins (ETs) (which are considered to be potent proinflammatory mediators) and LPC/ox-LDL, we investigated the interaction between ETs and LPC/ox-LDL by fluorescence spectroscopy and western blotting. Tryptophan fluorescence measurements revealed ETs specifically interacted with LPC at concentrations that exceeded the critical micelle concentration (CMC). The tryptophan residue in ETs was not likely to be involved directly in the interaction between ETs and LPC micelles. Tryptophan fluorescence quenching revealed tryptophan residue in ETs where LPC concentrations were below the CMC may be buried deeply in the peptide or may interact with other amino acid residues, whereas tryptophan residue in ETs in the presence of LPC at concentrations exceeding the CMC was exposed outside of the peptide. Furthermore, ETs bind to ox-LDL in a concentration-dependent manner. These results strongly suggest that ox-LDL contains micelle-rich LPCs and that ETs specifically interact with the bioactive LPC micelles. Further study of the interaction between ETs and LPC micelles contained in ox-LDL will provide important information on the development and progression of many inflammatory diseases, including atherosclerosis.

Endothelin.

Endothelin-1 is the most potent vasoconstrictor agent currently identified, and it was originally isolated and characterized from the culture media of aortic endothelial cells. Two other isoforms, termed endothelin-2 and endothelin-3, were subsequently identified, along with structural homologues isolated from the venom of Actractapis engaddensis known as the sarafotoxins. In this review, we will discuss the basic science of endothelins, endothelin-converting enzymes, and endothelin receptors. Only concise background information pertinent to clinical physician is provided. Next we will describe the pathophysiological roles of endothelin-1 in pulmonary arterial hypertension, heart failure, systemic hypertension, and female malignancies, with emphasis on ovarian cancer. The potential intervention with pharmacological therapeutics will be succinctly summarized to highlight the exciting pre-clinical and clinical studies within the endothelin field. Of note is the rapid development of selective endothelin receptor antagonists, which has led to an explosion of research in the field.

Identification of an endothelin-converting enzyme-2-specific fluorigenic substrate and development of an in vitro and ex vivo enzymatic assay.

Endothelin-converting enzyme-2 (ECE-2) is a membrane-bound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast® libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH(2) (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k(cat)/K(m)) of 8.1 ± 0.9 × 10(3) M(-1) s(-1). Using this novel substrate, we also characterized the first potent (K(i) = 7.7 ± 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock-out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.

The discovery of endothelin: the power of bioassay and the role of serendipity in the discovery of endothelium-derived vasocative substances.

Significant discoveries in biology and medicine are rare. The progress in these fields is predominantly incremental, but sometimes new observations revolutionize the field by opening new directions in research for decades to come. Two cornerstone observations in the late 1970s and early 1980s are examples of such "revolutionary" events. The first, by Furchgott and Zawadzki, was the discovery of the "obligatory role of the endothelium in vasorelaxation by acetylcholine". The other, by Hickey and colleagues, was the first description and characterization of a vasoconstrictor polypeptide produced by endothelial cells in culture. Both of these observations were achieved by the application of bioassay and serendipity played an important role in each of them. They both represent starting points for rapid growth in research activity world-wide leading to the identification of EDRF as nitric oxide, and the polypeptide EDCF as endothelin a few years later. These early observations also raised interest and initiated intensive R&D activity in the pharma industry culminating in the regulatory approval and marketing of novel medicines treating human diseases. This review describes the events leading to the discovery and early characterization of the peptidergic endothelium-derived constrictor factor, and its purification, sequencing and naming it endothelin.

Endothelins and sarafotoxins: physiological regulation, receptor subtypes and transmembrane signaling.

The endothelins and sarafotoxins are two structurally related families of potent vasoactive peptides. Although the exact physiological function of these peptides is not known, the endothelins are probably involved in pathophysiological conditions such as hypertension and heart failure. This article will review the biochemistry of these peptides and their receptors, with special reference to proteolytic regulation, receptor subtypes and the G protein-linked phosphoinositide signal transduction pathway.

Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation.

Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.

Profile of past and current clinical trials involving endothelin receptor antagonists: the novel "-sentan" class of drug.

Since its initial characterization in 1988, over 18,236 papers, including 2,485 reviews, have been published in the endothelin (ET) field. Over this period, several generations of selective and mixed (dual) ET receptor antagonists (ERAs), from peptidic backbones to orally active potent (subnanomolar) small molecular compounds, have been developed. These agents have been studied in many experimental animal models of various pathological conditions (cardiovascular, respiratory, and neuro-immunological). Continued basic research has led to a better understanding of the complex interactions between the ET axis and other biologic systems in human pathophysiology. The first clinical trial involved patients with idiopathic pulmonary arterial hypertension and led to approval of bosentan (Tracleer) for use in the United States and Europe in 2002. Since then, bosentan, the only currently approved dual (mixed) ERA, has been used in numerous other clinical trials. In addition, more selective ET(A) receptor antagonists (ambrisentan, atrasentan, avosentan, clazosentan, darusentan, and sitaxsentan) are undergoing clinical trials. Here we outline the ERAs undergoing development and summarize the standing of completed and ongoing trials at the time of the Ninth International Conference on Endothelin and even thereafter. This review is intended to provide a useful reference for those interested in the current state of clinical trials involving ERAs, and to identify lessons that might apply to the design of future trials.

Primary structure of cat preproendothelin-2 and cat renal mRNA expression of preproendothelin-1 and preproendothelin-2 in naturally occurring renal failure.

Endothelin (ET)-1 is involved in the pathophysiology of various renal disorders, promoting renal cellular proliferation and extracellular matrix protein accumulation, and, thus, diminishing fundamental renal function, including filtration. To determine whether ET-1 and ET-2 play a role in feline chronic renal failure, we analyzed the messenger RNA (mRNA) expression of the prepro-ET (PPET )-1 and PPET-2 genes in affected cat kidney after molecular cloning of full-length PPET-2 complementary DNA (cDNA). Conceptual analysis of the primary structure of cat PPET-2 based on the cloned sequence demonstrated that the putative regions corresponding to a mature peptide and peptidase processing sites are present in cat PPET-2. Homology analysis showed that the similarity of the cat PPET-2 amino acid sequence with those from human, mouse, rat, rabbit, dog, ferret, cow, and horse was 73.0%, 68.6%, 69.1%, 76.4%, 81.2%, 83.1%, 76.3%, and 79.2%, respectively. Analysis of PPET-1 and PPET-2 mRNA in cat by reverse transcription polymerase chain reaction showed upregulated expression of both genes in kidneys affected by renal failure.

Effects of endothelin family on ANP secretion.

The endothelins (ET) peptide family consists of ET-1, ET-2, ET-3, and sarafotoxin (s6C, a snake venom) and their actions appears to be different among isoforms. The aim of this study was to compare the secretagogue effect of ET-1 on atrial natriuretic peptide (ANP) secretion with ET-3 and evaluate its physiological meaning. Isolated nonbeating atria from male Sprague-Dawley rats were used to evaluate stretch-activated ANP secretion in response to ET-1, ET-2, ET-3, and s6C. Changes in mean blood pressure (MAP) were measured during acute injection of ET-1 and ET-3 with and without natriuretic peptide receptor-A antagonist (A71915) in anesthetized rats. Changes in atrial volume induced by increased atrial pressure from o to 1, 2, 4, or 6cm H2O caused proportional increases in mechanically-stimulated extracellular fluid (ECF) translocation and stretch-activated ANP secretion. ET-1 (10nM) augmented basal and stretch-activated ANP secretion in terms of ECF translocation, which was blocked by the pretreatment with ETA receptor antagonist (BQ123, 1µM) but not by ETB receptor antagonist (BQ788, 1µM). ETA receptor antagonist itself suppressed stretch-activated ANP secretion. As compared to ET-1- induced ANP secretion (3.2-fold by 10nM), the secretagogue effects of ANP secretion by ET-2 was similar (2.8-fold by 10nM) and ET-3 and s6C were less potent (1.7-fold and 1.5-fold by 100nM, respectively). Acute injection of ET-1 or ET-3 increased mean blood pressure (MAP), which was augmented in the presence of natriuretic peptide receptor-A antagonist. Therefore, we suggest that the order of secretagogue effect of ET family on ANP secretion was ET-1≥ET-2>>ET-3>s6C and ET-1-induced ANP secretion negatively regulates the pressor effect of ET-1.

Sequence-directed recognition peptides: inhibition of endothelin generation via a substrate-depletion mechanism.

Sequence-directed recognition peptides (SDRPs) were constructed on the basis of their hydropathic complementarity for big-endothelin (bigET). These peptides can inhibit in vitro the proteolytic cleavage that generates endothelin (ET) from its bigET precursor. Comparison of dissociation constants of the complexes SDRP:bigET with kinetic constants obtained for the cleavage of bigET by alpha-chymotrypsin (taken as a model proteinase) provides evidence of the potential of SDRPs. This is a novel application of SDRPs used as inhibitors of a proteolytic reaction.

Endothelin receptor agonists and antagonists exhibit different dissociation characteristics.

Endothelins (ETs) are vasoconstricting peptides that bind to membrane receptors to initiate their physiological effects. This report compares the dissociation characteristics of selected ET agonists and antagonists, and studies the effects of any difference in dissociation characteristics on the potency of antagonists. Competition studies using various ET receptor ligands against [125I]ET-1 or [125I]ET-3 binding demonstrated that porcine cerebellum membranes contain predominantly ETB receptor. [125I]IRL1620 associated with the receptors in a time-dependent manner. Although bound [125I]IRL1620 was easier to dissociate than bound [125I]ET-3, both agonists exhibited a dissociation half life > 20 h. For non-radiolabeled ligands, bind-and-wash studies were employed in which membranes were pre-incubated with unlabeled ligand followed by extensive washing before assaying for [125I]ET-1 binding. Results from bind-and-wash studies confirmed that bound non-radiolabeled IRL1620 and ET were as difficult to dissociate as [125I]ligands. In contrast, bound PD142893 and Ro46-2005 were easily dissociated from ETB receptors. Consequently, the inhibitory effects of PD142893 and Ro46-2005 on [125I]agonist binding diminished following incubation time. In cloned human ETA and ETB receptors, bound ET-1 was also more difficult to dissociate than bound antagonists. These results suggest that the differences in the dissociation characteristics of ET receptor agonists vs. antagonists may account for the diminished potency of Ro46-2005 and PD142893 as a function of incubation time.

Nucleotide sequence of endothelin-1 cDNA from rabbit endothelial cells.

A cDNA encoding rabbit endothelin-1 (ET-1) was isolated by plaque hybridization from a rabbit inferior vena caval endothelial cell lambda gt11 cDNA library using human ET-1 cDNA as the hybridization probe. DNA sequence analysis indicates that mature 21 amino acid rabbit ET-1 is derived from a 202 amino acid precursor, via a 39 amino acid intermediate 'big' ET-1.

Molecular characterization of endothelin receptors.

Following the first report on the identification of endothelin (ET), an increasing body of work has accumulated on this endothelium-derived 21-amino acid vasoconstrictor peptide. Subsequently, the existence of three distinct isoforms of ET, designated ET-1, ET-2 and ET-3, was predicted from the finding of three separate genes. The differential potencies of the three isoforms of the ET family have opened up the possibility of the existence of multiple ET receptor subtypes. Recently, molecular biological techniques provided direct evidence of at least two distinct subtypes of ET receptor. This article discusses the functions of the ETs, focusing especially on the molecular characteristics of their receptors.

Endothelins and sarafotoxins: physiological regulation, receptor subtypes and transmembrane signaling.

The endothelins and sarafotoxins are two structurally related families of potent vasoactive peptides. Although the physiological functions of these peptides are not entirely clear, the endothelins are probably involved in pathophysiological conditions such as hypertension and heart failure. This review summarizes the state of the art in some areas of this intensively studied subject, including: (1) structure-function relationships of ET/SRTX, (2) ET concentrations in plasma, (3) ET/SRTX receptor subtypes and (4) signaling events mediated by the activation of ET/SRTX receptors.

The structure and specificity of endothelin receptors: their importance in physiology and medicine.

In addition to involvement in vascular endothelium-smooth muscle communication, the secretion of and receptors for, endothelins are widely distributed. Two cloned receptor subtypes are G-protein-coupled to several intracellular messengers, predominantly inositol phosphates. From a knowledge of structure-activity relationships and peptide conformations, details of receptor architecture and selective agents, including nonpeptides and antagonists, have been discovered. From the nature of the actions of endothelins, receptor distributions (including CNS) and plasma levels, it is concluded that they are paracrine factors normally involved in long-term cellular regulation, but which may be important in several pathologies, many of which are stress-related.

Endothelin receptor antagonists: an overview of their synthesis and structure-activity relationship.

Endothelins (ETs) are potent vasoconstrictor peptides and are associated with several disease states like pulmonary hypertension, systemic hypertension and heart failure. Endothelin-1 (ET-1) is the first member of the family and it has the receptor subtypes known as ETA and ETB. The receptors ETA and ETB are attractive new therapeutic targets for diseases associated with elevated ET-1 levels. Several studies have thus led to the discovery of selective ETA receptor antagonists as well as non-selective ETA/ETB antagonists. The preclinical and clinical studies have clearly established that these antagonists are effective in the treatment of essential hypertension, pulmonary hypertension, heart failure and atherosclerosis. The advances in this area have resulted in the FDA approval of the orally active dual antagonist Bosentan for pulmonary hypertension in 2001. This review highlights the synthesis and structure-activity of the endothelin receptor antagonists and covers the literature in this area up to 2001.

Proteolytic processing pattern of the endothelin-1 precursor in vivo.

Endothelin-1 (ET-1) is a potent vasoconstrictor, which has been implicated in diseases involving dysfunctions of the cardiovascular system. For the biogenesis of ET-1, a larger precursor peptide (proET-1) is cleaved at two sites to give rise to bigET-1, which is subsequently cleaved to generate mature ET-1. In the present study, we investigated, which other peptides are derived from proET-1 in vivo. Six sandwich immunoassays covering various regions of proET-1 were developed and used to detect circulating proET-1 immunoreactivities in plasma of healthy subjects and septic patients. With this approach we could (a) demonstrate that, in addition to bigET-1/ET-1, three stable proET-1 fragments are generated, (b) exclude two previously discussed regions as sites for prohormone conversion and (c) show that the proteolytic processing pattern of proET-1 is unchanged under pathological conditions, which are associated with elevated levels of proET-1 fragments. The high stability and similarity in concentration of the proET-1 fragments suggest that these might be non-functional in the circulation. Stable proET-1 fragments maybe used in the future as reliable diagnostic targets to indirectly assess the release of ET-1, which might help to more selectively direct therapeutic measures.

Advances in Vascular Hyporeactivity After Shock: The Mechanisms and Managements.

Vascular reactivity to vasoconstrictors and vasodilators is greatly reduced after severe trauma, shock, and sepsis or multiple organ dysfunction syndrome. This reduced vascular reactivity severely interferes with the treatment of shock and other critical conditions. In particular, it interferes with the efficacy of vasoactive agents. Consequently, it is very important to elucidate the mechanisms and search for the effective treatment measures. In recent years, a lot of studies focused on the characteristics and the change rules of vascular hyporeactivity and mechanisms following shock. Also, the treatment approaches based on various mechanisms have been a hot pot these years.