Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.     

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.     

Molecular and functional evidence for early divergence of an endothelin-like system during metazoan evolution: analysis of the Cnidarian, hydra

A novel putative endothelin-converting enzyme (ECE) has been cloned from hydra, a freshwater invertebrate that belongs to the second oldest phylum of the animal kingdom. As an integral component of the endothelin system, vertebrate ECE functions in the activation of endothelin (ET) peptides. Vertebrate ETs are (1) the most potent vasoconstrictors known in mammals; and (2) function as essential signaling ligands during development of tissues derived from neural crest cells. To date, only a limited number of immunocytochemical studies have suggested the presence of endothelin-like peptides in invertebrates. Based on structural and functional analyses, we present evidence for a functional endothelin-like system in hydra that is involved in both muscle contraction and developmental processes. These findings indicate the broad use of endothelin systems in metazoans and also indicate that this type of signaling system arose early in evolution even before divergence of protostomes and deuterostomes.     

Endothelin converting enzyme-2: a processing enzyme involved in the generation of novel neuropeptides

Members of several metalloprotease families have been proposed to be involved in non-classical processing of neuroendocrine precursors. Among them, endothelin converting enzyme-2 (ECE-2) is a good candidate since it exhibits a neuroendocrine distribution, intracellular subcellular localization, and an acidic pH optimum. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. These results are consistent with an important role for ECE-2 in the processing of regulatory peptides at non-classical sites.     

Purification and characterization of putative endothelin converting enzyme in bovine adrenal medulla: evidence for a cathepsin D-like enzyme

A specific and sensitive assay has been established for measurement of endothelin converting activity in a tissue extract. This assay is based on measuring endothelin-1 generated from big endothelin-1 by endothelin converting enzyme (ECE) with radioimmunoassay using an endothelin C-terminal specific antibody. By using this assay, we purified and characterized ECE in bovine adrenomedullary chromaffin granules ECE was purified over 3,000 times by a combination of DEAE, hydrophobic and gel filtration chromatography. A molecular weight of ECE was estimated to be approximately 30,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ECE had three major components with estimated molecular weights of 45,000, 30,000 and 15,000 like bovine spleen cathepsin D. ECE had a pH optimum at 3.5 and was inhibited by pepstatin. These results strongly suggest that ECE is a cathepsin D-like aspartic protease.     

Applicability of green fluorescence protein in the study of endothelin converting enzyme‐1c trafficking

Endothelin‐1 (ET‐1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor big endothelin‐1 by endothelin converting enzyme‐1 (ECE‐1). Production of ET‐1 is thought to be dependent upon the expression of ECE‐1 at the cell surface. Therefore, mechanisms inducing the trafficking of ECE‐1 to the cell surface have been the focus of recent research. This research has identified phosphorylation of the cytoplasmic region of ECE‐1 as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE‐1 to monitor phosphorylation induced trafficking of ECE‐1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE‐1, and hence the suitability of GFP or any other protein tag in studying ECE‐1 distribution and trafficking. ECE‐1c is the most widely expressed isoform in endothelial cells. We therefore expressed ECE‐1c with a GFP tag either at the N or C‐terminus of ECE‐1c. Catalytic activity and effect on protein kinase C (PKC) induced phosphorylation was compared between the two chimeras and wild‐type ECE‐1c. Our results indicate that positioning of the GFP tag on the C‐terminus abrogates activity without effecting PKC‐induced phosphorylation. However, GFP tag on the N‐terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE‐1c distribution and trafficking.     

The Endothelin System and Endothelin-Converting Enzyme in the Brain: Molecular and Cellular Studies

The biologically active vasoactive peptides, the endothelins (ETs), are generated from inactive intermediates, the big endothelins, by a unique processing event catalysed by the zinc metalloprotease, endothelin converting enzyme (ECE). In this overview we examine the actions of endothelins in the brain, and focus on the structure and cellular locations of ECE. The heterogeneous distribution in the brain of ET-1, ET-2, and ET-3 is discussed in relation to their hemodynamic, mitogenic and proliferative properties as well as their possible roles as neurotransmitters. The cellular and subcellular localization of ECE in neuronal and in glial cells is compared with that of other brain membrane metalloproteases, neutral endopeptidase-24.11 (neprilysin), angiotensin converting enzyme and aminopeptidase N, which all function in neuropeptide processing and metabolism. Unlike these ectoenzymes, ECE exhibits a dual localisation in the cell, being present on the plasma membrane and also, in some instances, being concentrated in a perinuclear region. This differential localization may reflect distinct targeting of different ECE isoforms, ECE-l, ECE-1, and ECE-2.     

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.     

CGS 34043: a non-peptidic, potent and long-acting dual inhibitor of endothelin converting enzyme-1 and neutral endopeptidase 24.11

Endothelin-1 (ET- 1) is a potent vasoconstrictor. Its biosynthesis is catalyzed by endothelin converting enzyme (ECE). In contrast, atrial natriuretic peptide (ANP) is a potent vasorelaxant and diuretic, and it is mainly degraded by neutral endopeptidase 24.11 (NEP). Therefore, compounds that can suppress the production of ET-1 by inhibiting ECE while simultaneously potentiating the levels of ANP by inhibiting NEP may be novel agents for the treatment of cardiovascular and renal dysfunction. CGS 34043 is one such compound, which inhibited the activities of ECE-1a and NEP with IC50 values of 5.8 and 110 nM, respectively. In vivo, it inhibited the pressor response induced by big ET-1, the precursor of ET-1, dose-dependently in rats, and the inhibition was sustained for at least 2 hr. In addition, CGS 34043 increased plasma ANP by 150% up to 4 hr after an intravenous dose of 10 mg/kg in conscious rats infused with ANP. However, this compound had no effect on the angiotensin I-induced pressor response. These results demonstrate that CGS 34043 is a potent and long-lasting dual inhibitor of ECE-1 and NEP. Consequently, it may be beneficial for the treatment of diseases in which an overproduction of ET-1 and/or enhanced degradation of ANP plays a pathogenic role. The activity of CGS 34753, an orally active prodrug of CGS 34043, is also described.     

Nonpeptidic endothelin-converting enzyme inhibitors and their potential therapeutic applications

Endothelins (ETs) are potent vasoconstrictors, promitogens, and inflammatory mediators. They have been implicated in the pathogenesis of various cardiovascular, renal, pulmonary, and central nervous system diseases. Since the final step of the biosynthesis of ETs is catalyzed by a family of endothelin-converting enzymes (ECEs), inhibitors of these enzymes may represent novel therapeutic agents. Currently, seven isoforms of these metalloproteases have been identified; they all share a significant amino acid sequence identity with neutral endopeptidase 24.11 (NEP), another metalloprotease. Therefore, it is not surprising that the majority of ECE inhibitors also possess potent NEP inhibitory activity. To date, three classes of ECE inhibitors have been synthesized: dual ECE/NEP inhibitors, triple ECE/NEP/ACE inhibitors, and selective ECE inhibitors. Potential clinical applications of these compounds in hypertension, chronic heart failure, restenosis, renal failure, and cerebral vasospasm deduced from studies with relevant animal models are reviewed.     

Functional evidence for the presence of a phosphoramidon-sensitive enzyme in rat brain that converts big endothelin-1 to endothelin-1

Endothelin-1 (ET-1) is produced from its precursor, big endothelin-1 (BigET-1), by a putative endothelin-converting enzyme (ECE), but it is not known whether the enzyme is present in the brain. This study was conducted to examine the central hemodynamic effects of BigET-1 and to indirectly determine the presence of an ECE in rat brain. Cardiovascular effects of centrally administered BigET-1 and ET-1 were examined in anesthetized, ventilated rats. BigET-1 (100 pmol) or ET-1 (10 pmol) applied to the IV ventricle produced similar prolonged decreases in mean arterial pressure (MAP) and renal blood flow (RBF). Thus, peak decreases with BigET-1 were (mean +/- S.E.): MAP = -35 +/- 4%; RBF = -27 +/- 5%, while those with ET-1 were: MAP = -36 +/- 5%; RBF = -29 +/- 9%. Pretreatment with phosphoramidon, a metalloprotease inhibitor (90 nmol), abolished the hemodynamic responses elicited by BigET-1 (MAP = -9 +/- 2%; RBF = -3 +/- 2%) but not those produced by ET-1. These data indicate that; i) conversion of BigET-1 to ET-1 in the brain is essential for the expression of hemodynamic actions and ii) a metalloprotease capable of converting BigET-1 to ET-1 is present in rat brain.     

Animals lacking endothelin-converting enzyme-2 are deficient in learning and memory

Endothelin-converting enzyme (ECE)-2 is a metalloprotease that possesses many properties consistent with it being a neuropeptide-processing enzyme. This protease is found primarily in neural tissues, with high levels of expression in midbrain, cerebellum, hypothalamus, frontal cortex and spinal cord and moderate levels in hippocampus and striatum. To evaluate its role in neural function, mice have been generated lacking this enzyme. Physical appearance, autonomic reflexes, motor co-ordination, balance, locomotor activity and spontaneous emotional responses appear normal in these knockout (KO) mice. However, these mutants display deficits in learning and memory as evidenced by marked impairment in the Morris water maze. Knockout mice are also deficient in object recognition memory where they show delays in discerning changes in object location and in recognizing the introduction of a novel object. In this study, perseveration appears to interfere with learning and memory. Finally, mutants are impaired in social transmission of food preference where they show poor short-term memory and perturbations in long-term memory; the latter can be ameliorated by reminder cues. As ECE-2 has been implicated in Alzheimer's disease, the deficits in learning and memory in the KO mice may provide unique insights into processes that may contribute to this disease and possible other disorders of cognition.     

Zinc peptidases and Alzheimer's disease

We have investigated the role of zinc peptidases in metabolism of the amyloid precursor protein (APP) and the effects of hypoxia. Two peptidase families have been studied: the neprilysin (NEP) family which includes, in the brain: NEP, endothelin converting enzyme (ECE) and secreted endopeptidase (SEP). Reactive oxygen species can regulate enzyme activity via modulation of the zinc ion at the active site. Both NEP and ECE can prevent accumulation of amyloid beta peptide by hydrolyzing the peptide. As acute and chronic hypoxia can modulate APP processing, we have investigated the effects of hypoxia in cell culture on the expression and activity of NEP, ECE and SEP. In parallel, we have monitored the expression of another zinc peptidase, alpha-secretase, that mediates the nonamyloidogenic processing of APP. Overall, zinc peptidases appear neuroprotective and modulation of these activities in pathological states could lead to neurodegeneration.
Acknowledgements:  This work was supported by the UK MRC, The Royal Society, INTAS, The Biochemical Society.     

Partial purification of phosphoramidon-sensitive endothelin converting enzyme in porcine aortic endothelial cells: high affinity for Ricinus communis agglutinin.

A membrane-bound endothelin converting enzyme (ECE) of porcine aortic endothelial cells (ECs) was solubilized with Lubrol PX with high efficiency and stability. The solubilized ECE was bound to Ricinus communis agglutinin (RCA) but not to peanut agglutinin (PNA) or wheat germ agglutinin (WGA), suggesting that the ECE has a galactosylated structure possessing a high affinity for RCA. The sequential chromatography on RCA-agarose, PNA-agarose and a TSKgel DEAE-5PW column attained 2,100-fold purification for the ECE over the membrane fractions. The purified ECE was sensitively inhibited by phosphoramidon but not by thiorphan. The present RCA and PNA affinity column procedures may be a powerful approach to isolation of ECE of EC origin.     

Identification and partial purification of a thiol endothelin-converting enzyme from porcine aortic endothelial cells.

Endothelin is a potent peptide vasoconstrictor. The final step in the processing of endothelin has been postulated to be the cleavage of the Trp21-Val22 peptide bond in proendothelin by a putative endothelin-converting enzyme. A soluble extract of primary porcine aortic endothelial cells was found to contain an enzyme activity that converted proendothelin-1 (proET-1) to an endothelin-1 (ET-1)-like peptide as determined by the rabbit aortic ring contraction assay. This enzyme was partially purified by DE52 ion-exchange chromatography. Incubation of proET-1 with the partially purified enzyme generated a product which had a retention time on HPLC identical to that of authentic ET-1. Further analysis of the product showed that it caused contraction of rabbit aortic rings, had a molecular weight identical to ET-1 as measured by fast atom bombardment mass spectrometry, and competed for [125I]ET-1 binding in an RIA using specific antibodies which recognize the carboxy terminal tryptophan of ET-1. The enzyme activity could be inhibited by thiol protease inhibitors such as Z-phe-pheCHN2 and p-hydroxymercuribenzoate, but not by serine- or metalloprotease inhibitors. The optimal pH for the enzymatic activity was between 7.0 and 7.5, and no activity was detected at pH 4.0. These results demonstrate that this thiol protease is a potential endothelin-converting enzyme.     

Superoxide regulation of endothelin-converting enzyme

Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.     

A high molecular weight metalloendoprotease from the cytosol of mammalian cells

In red cell lysates, three soluble proteases hydrolyze insulin at pH 8.5. One of these enzymes was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA, o-phenanthroline, and 8-hydroxyquinoline, the metal-depleted enzyme can be reactivated by micromolar levels of Zn2+, Co2+, or Mn2+, and it is not inhibited by reagents specific for carboxyl, serine or thiol proteases. This enzyme has an apparent molecular weight of 300,000 +/- 25,000, and electrophoresis in sodium dodecyl sulfate indicates a single band with an Mr = 115,000 +/- 10,000. End group analysis and automated Edman degradation of the products of proteolysis showed that it is an endoprotease which cleaves on the NH2-terminal side of large hydrophobic amino acids. Although various small polypeptides with Mr = 2300-3500 are hydrolyzed (e.g. insulin chains, glucagon, and calcitonin), a variety of larger proteins are not degraded (e.g. casein and globin). The latter proteins, however, are converted to substrates for the metalloprotease by digestion with the ATP-stimulated endoprotease from erythrocytes. Thus, the metalloprotease may play a role in the ATP-dependent pathway for degrading proteins with abnormal structures and could account in part for the o-phenanthroline sensitivity of this process. A similar enzyme is found in humans, rabbits, and rats and is cytosolic in all tissues which have been examined including erythrocytes, reticulocytes, liver, kidney, brain, and skeletal muscle.     

The effects of novel cathepsin E inhibitors on the big endothelin pressor response in conscious rats.

The aspartic protease, cathepsin E, has been shown to specifically cleave big endothelin (big ET-1) at the Trp21-Val22 bond to produce endothelin (ET-1) and the corresponding C-terminal fragment. To determine whether cathepsin E is a physiologically relevant endothelin converting enzyme (ECE), three novel and potent inhibitors of cathepsin E were administered to conscious rats prior to a pressor challenge with big ET-1. One of the inhibitors of cathepsin E, SQ 32,056 (3 mg/kg i.v.), blocked the big ET-1 response. However, this dose of SQ 32,056 also blocked the pressor response to ET-1. Phosphoramidon specifically inhibited the Big ET-1 pressor response. These results suggest that ECE is not cathepsin E.     

Expression of Endothelin-Converting Enzyme in Both Neuroblastoma and Glial Cell Lines and Its Localization in Rat Hippocampus

Abstract: Endothelin-converting enzyme is a phosphoramidon-sensitive metalloprotease that cleaves big endothelin to the potent vasoconstrictor peptide, endothelin. The converting enzyme is expressed in endothelial cells in a variety of tissues and in some secretory cells. In the present study, phosphoramidon-sensitive endothelin-converting enzyme activity has been demonstrated by radioimmunoassay in the neuroblastoma cell line, SH-SY5Y, and in Bu17 and C6 glioma lines. The identity of the activity was confirmed by immunoblotting, revealing a polypeptide of ∼120 kDa in each of these lines, in D384 glioma cells, and in primary astrocytes. Immunofluorescence revealed the cell-surface location of endothelin-converting enzyme in the neuronal and glial cell lines and in primary astrocytes. Pretreatment of SH-SY5Y and Bu17 cells with phosphoramidon resulted in an apparent concentration of the enzyme protein in an intracellular compartment. Immunoperoxidase-staining of rat brain sections located this metalloprotease to the pyramidal cells of the hippocampus. Endothelin-converting enzyme-1 was revealed by in situ hybridisation in the neuronal and glial cell lines.     

Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells. Analysis of endothelin-2 biosynthetic pathway.

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.