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.

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.

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.

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.

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.

The Second Tomoh Masaki Award (2013).

Professor Katsutoshi Goto, phD, a Japanese physio-pharmacologist born in 1943, is the recipient of the The Second Tomoh Masaki Award, a bi-annual prize presented on the occasion of the International Conferences on Endothelin to scientists for outstanding contributions and achievements in the field of endothelin research. The Second Tomoh Masaki Award was presented to Professor Goto at the Thirteenth International Conference on Endothelin held at Tokyo Campus of the University of Tsukuba, Tokyo, Japan, in September 2013 in recognition of his scientific contributions in the 1980s that ultimately allowed to identify and characterize the endothelin peptides. Goto's innovative work was quintessential in the discovery of endothelin which has led to the development of new therapies and new clinical applications in medicine and particularly pulmonary arterial hypertension. This article summarizes the career and the scientific achievements of Katsutoshi Goto, it includes statements of former students on his role as a mentor as well as the awardee's personal quotes, including a message to young endothelin researchers.

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.

Non-enzymatic proteins from snake venoms: a gold mine of pharmacological tools and drug leads.

Non-enzymatic proteins from snake venoms play important roles in the immobilization of prey, and include some large and well-recognized families of toxins. The study of such proteins has expanded not only our understanding of venom toxicity, but also the knowledge of normal and disease states in human physiology. In many cases their characterization has led to the development of powerful research tools, diagnostic techniques, and pharmaceutical drugs. They have further yielded basic understanding of protein structure-function relationships. Therefore a number of studies on these non-enzymatic proteins had major impact on several life science and medical fields. They have led to life-saving therapeutics, the Nobel prize, and development of molecular scalpels for elucidation of ion channel function, vasoconstriction, complement system activity, platelet aggregation, blood coagulation, signal transduction, and blood pressure regulation. Here, we identify research papers that have had significant impact on the life sciences. We discuss how these findings have changed the course of science, and have also included the personal recollections of the original authors of these studies. We expect that this review will provide impetus for even further exciting research on novel toxins yet to be discovered.

Endothelin systems in the brain: involvement in pathophysiological responses of damaged nerve tissues.

In addition to their potent vasoconstriction effects, endothelins (ETs) show multiple actions in various tissues including the brain. The brain contains high levels of ETs, and their production is stimulated in many brain disorders. Accumulating evidence indicates that activation of brain ET receptors is involved in several pathophysiological responses in damaged brains. In this article, the roles of brain ET systems in relation to brain disorders are reviewed. In the acute phase of stroke, prolonged vasospasm of cerebral arteries and brain edema occur, both of which aggravate brain damage. Studies using ET antagonists show that activation of ETA receptors in the brain vascular smooth muscle induces vasospasm after stroke. Brain edema is induced by increased activity of vascular permeability factors, such as vascular endothelial growth factor and matrix metalloproteinases. Activation of ETB receptors stimulates astrocytic production of these permeability factors. Increases in reactive astrocytes are observed in neurodegenerative diseases and in the chronic phase of stroke, where they facilitate the repair of damaged nerve tissues by releasing neurotrophic factors. ETs promote the induction of reactive astrocytes through ETB receptors. ETs also stimulate the production of astrocytic neurotrophic factors. Recent studies have shown high expression of ETB receptors in neural progenitors. Activation of ETB receptors in neural progenitors promotes their proliferation and migration, suggesting roles for ETB receptors in neurogenesis. Much effort has been invested in the pursuit of novel drugs to induce protection or repair of damaged nerve tissues. From these studies, the pharmacological significance of brain ET systems as a possible target of neuroprotective drugs is anticipated.

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.

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.

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.

Thyme extract, but not thymol, inhibits endothelin-induced contractions of isolated rat trachea.

Thyme extract is known to posses spasmolytic effects on isolated trachea and to increase ciliary activity. A small part of this effect is mediated via beta (2) receptors, but other receptors are supposed to be involved. Endothelin has a pathophysiological impact in asthma with respect to hyper-reagibility and contracting isolated trachea smooth muscles and was, therefore, investigated. Thyme extract inhibited endothelin-induced contraction. Emax decreased depending on the concentration of thyme extract indicating a non-competitive inhibition. Bosentan, an endothelin receptor antagonist (positive control), acted as a competitive inhibitor. The interaction/combination of thyme extract and bosentan was additive but not supra-additive indicating an interaction with the endothelin system. Thymol (one of the major compounds of thyme extract) did not interact with the endothelin system. It is concluded that thyme extract may help in diseases related to endothelin hyper-reagibility of the bronchus system such as asthma and COPD (chronic obstructive pulmonary disease), although thymol is not involved in this effect.

Endothelin and endothelin converting enzyme-1 in the fish gill: evolutionary and physiological perspectives.

In euryhaline fishes like the killifish (Fundulus heteroclitus) that experience daily fluctuations in environmental salinity, endothelin 1 (EDN1) may be an important regulator molecule necessary to maintain ion homeostasis. The purpose of this study was to determine if EDN1 and the endothelin converting enzyme (ECE1; the enzyme necessary for cleaving the precursor proendothelin-1 to EDN1) are present in the killifish, to determine if environmental salinity regulates their expression, and to examine the phylogenetic relationships among the EDNs and among the ECEs. We sequenced killifish gill cDNA for two EDN1 orthologues, EDN1A and EDN1B, and also sequenced a portion of ECE1 cDNA. EDN1A and ECE1 mRNA are expressed ubiquitously in the killifish while EDN1B mRNA has little expression in the killifish opercular epithelium or gill. Using in situ hybridization and immunohistochemistry, EDN1 was localized to large round cells adjacent to the mitochondrion-rich cells of the killifish gill, and to lamellar pillar cells. In the gill, EDN1A and EDN1B mRNA levels did not differ with acute (<24 h) or chronic (30 days) acclimation to seawater (SW); however, EDN1B levels increased threefold post SW to freshwater (FW) transfer, and ECE1 mRNA levels significantly increased twofold over this period. ECE1 mRNA levels also increased sixfold over 24 h post FW to SW transfer. Chronic exposure to SW or FW had little effect on ECE1 mRNA levels. Based upon our cellular localization studies, we modeled EDN1 expression in the fish gill and conclude that it is positioned to act as a paracrine regulator of gill functions in euryhaline fishes. It also may function as an autocrine on pillar cells, where it is hypothesized to regulate local blood flow in the lamellae. From our phylogenetic analyses, ECE is predicted to have an ancient origin and may be a generalist endoprotease in non-vertebrate organisms, while EDNs are vertebrate-specific peptides and may be key characters in vertebrate evolution.

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.

Endothelins and airways--a short review.

Endothelins (ETs) are a multifunctional large family of polypeptides. There are three well recognized members in this family (ET-1, ET-2, and ET-3) of which ET-1 appears to be the most important. They have been shown to play an important role in the pathogenesis of many life threatening diseases of humans and animals. They also perform a wide variety of physiological roles. The most important property of ETs is smooth muscle contraction, which allows them to play an important role in the pathogenesis of many vascular, gastrointestinal, urogenital and airway diseases. Another important feature of ETs is their influence on the immune system. Many animal and human studies have shown that antagonists of ET receptors can remarkably alleviate many disease symptoms. ETs produce their effect by acting via two established types of receptors namely ET-A and ET-B, which are present in various type of cells in the body. These receptors have varied and sometimes opposite functions. Pulmonary vascular endothelium is the richest source of ET in the body. Lung is the primary organ of ET metabolism and clearance. It has been reported that ETs play a pivotal role in the pathogenesis of asthma, chronic obstructive pulmonary disease, bronchiolitis obliterans and other important airway diseases. Many of these obstructive airway diseases are characterized by bronchoconstriction, mucous hyperplasia, airway remodeling and inflammation. ET is involved in all of these symptoms. In spite of its involvement in many diseases, the exact role of ET in the pathogenesis of these diseases remains unclear. The purpose of this review is to give the reader an insight regarding the importance of multitude and diverse roles played by ETs in various airway diseases.

Endothelin-like peptides.

Mammalian endothelins (ETs) and snake venom sarafotoxins (SRTXs) comprise structurally and functionally related potent vasoconstrictor isopeptides that act on the vascular system via identical receptors. This similarity is remarkable, since SRTXs are highly toxic components isolated from the venoms of snakes of the genus Atractaspis of the Atractaspididae family, while ETs are endogenous hormones of the mammalian vascular system. Since the first functional and structural description of SRTXs in 1988, the full extent of their natural diversity has become increasingly apparent, and this has led to the characterization of new families of endothelin-like peptides. Based on a combination of conventional biochemical approaches and the latest molecular biology and mass spectrometry techniques, this review describes the more recent panel of SRTX isopeptides isolated from various snake species within the Atractaspididae family, but also the similarities and differences that exist between sarafotoxins and endothelins in terms of their metabolism, genetic origin, structure and functional sites.

Characterization of toxins within crude venoms by combined use of Fourier transform mass spectrometry and cloning.

The standard analytical procedure for screening the proteomic profile of a venom often relies on an appropriate combination of sample extraction, electrophoresis, reversed-phase high-performance liquid chromatography, mass spectrometry, and Edman degradation. We present in this study a new approach for venom screening based on Fourier transform mass spectrometry (FTMS) analysis directly on the crude venom. The venom chosen is a unique sample from Atractaspis irregularis, a species never studied at the molecular level previously. This snake belongs to the Atractaspidae family that is known to produce highly toxic venoms containing endothelin-like peptides called sarafotoxins (SRTXs). Nanoelectrospray-FTMS spectrum of the crude venom allowed the identification of 60 distinct compounds with molecular masses from 600 to 14,000 Da, which would have been impossible without the resolution of this kind of instrument. De novo sequencing within the entire venom confirmed the sequences of two new families of sarafotoxins, whose precursors had been cloned, and allowed the characterization of a third one. One particularly interesting point was that the propolypeptides appeared processed not in one unique compound, but rather in different length molecules ranging from 15 for the shorter to 30 amino acids for the longer. Moreover, our results clearly establish that in the case of A. irregularis only one copy of mature sarafotoxin emerges from each precursor, which is a totally different organization in comparison of other precursors of SRTXs.