Big ET-1 Research Articles

Identification of amino acid residues in the C-terminal tail of big endothelin-1 involved in processing to endothelin-1.

Big endothelin-1 (big ET-1) is converted to 21-amino acid residue endothelin-1 (ET-1) via a specific cleavage at Trp21-Val22 by endothelin converting enzyme (ECE). This conversion is an essential step to produce bioactive ET-1 and represents a regulatory site in the biosynthesis of this potent vasoconstrictor. ECE-1a, a unique membrane-bound enzyme, processes big ET-1 more efficiently than other big ET isoforms, which mainly differ in the C-terminal tail (residues 22-38). In this study, each of the highly conserved residues, Val22, Pro25, Pro30, Gly32, Leu33, and Gly34 were replaced with Ala in the preproendothelin-1 (PPET-1) cDNA using site-directed mutagenesis. The mutant and wild-type cDNAs were transiently transfected into Chinese hamster ovary cells along with ECE-1a cDNAs, and concentrations of the resulting recombinant peptides, ET-1 and big ET-1, in the transfection media were then measured. The concentration of immunoreactive ET-1 in the media from Val22, Pro25, Pro30, Gly32, and Leu33 mutant PPET-1-transfected cells was 4- to 6-fold lower than that of wild type and (Gly34-->Ala)PPET-1. Moreover, with the exception of Gly34 there was a corresponding increase in the concentrations of immunoreactive big ET-1 in the media from mutants. Similar results were obtained when His27, Val28, and Ser35 of big ET-1( )were substituted with the corresponding residues in big ET-2 and big ET-3. These findings suggest that the C-terminal tail has an important role in the intracellular processing of big ET-1 by ECE-1a. Herein we also report that a recombinant big ET-1 analog we previously generated and characterized, (Ala21)big ET-1, inhibits ECE-1a activity in a dose-dependent (K i=1 microM) and competitive manner.

Soluble Human Endothelin-Converting Enzyme-1: Expression, Purification, and Demonstration of Pronounced pH Sensitivity

Endothelin-converting enzyme-1 (ECE-1) is a type II integral membrane protein that belongs to a family of metalloproteases which includes ECE-2, neprilysin (neutral endopeptidase 24.11, EC 3.4.24.11), and Kell blood group protein. ECE-1 cleaves its biologically inactive native substrate, big endothelin-1, to generate a powerful vasoactive 21-amino acid peptide, endothelin-1. ECE-1 consists of a short N-terminal cytoplasmic tail, a transmembrane hydrophobic domain, and a large extracellular domain containing the catalytic site with a conserved Zn-binding motif. We have constructed a secreted, soluble form of ECE-1 (solECE-1) by fusing the cleavable N-terminal signal sequence of human alkaline phosphatase in frame with the entire extracellular domain of ECE-1. Stable transfectant CHO cell lines expressing up to 6.1 mg of solECE-1 per liter culture medium were established and solECE-1 was purified to homogeneity using three chromatographic steps with a 24% yield. SolECE-1 behaves as a dimer of 110-kDa subunits. SolECE-1 has a sharp pH optimum, similar to the native form, ECE-1a, but has a slightly more acidic pH optimum of 6.1–6.4 than that of 6.7–6.9 for ECE-1a. At its optimal pH of 6.4, solECE-1 cleaved big ET-1:big ET-2:big ET-3 in a ratio of 8.1:1:1.4, was inhibited by phosphoramidon with an IC50value of 0.35 ± 0.05 μM, had aKmvalue of 4.65 ± 0.78 μM for big ET-1, and had akcatvalue of 5.82 ± 0.21 min−1, all values comparable to those for ECE-1a at its optimal pH of 6.8. Phosphoramidon inhibition of both ECE-1a and solECE-1 is highly pH-dependent. At pH 5.8, phosphoramidon inhibited ECE-1a and solECE-1 with IC50values of 14 and 33 nM, respectively, which are 49- and 1224-fold more potent than at pH 7.2. SolECE-1 is highly glycosylated, similar to ECE-1a. Deglycosylation of solECE-1 by peptide N-glycosidase F shifted the apparent molecular weight of solECE-1 to approximately 80 kDa and the deglycosylated form(s) of solECE-1 preserved at least 72% of the activity of the glycosylated form.

Endothelin blockers and renal protection: a new strategy to prevent end-organ damage in cardiovascular disease?

Time for primary review 27 days. The endothelin (ET) family comprises three 21-amino acid peptides: ET-1, ET-2 and ET-3. Endothelin-1 has been identified as the major cardiovascular isopeptide. Release of the active peptide ET-1 requires cleavage of a Trp21–Val22 bond in the carboxyterminal of the precursor molecule, big ET-1 [1]. This reaction is catalyzed by a membrane bound metalloprotease, endothelin converting enzyme (ECE-1) [1, 2]. An intracellularly located ECE (ECE-2) has been identified as well [3]. Endothelin release is increased transcriptionally when the endothelium is exposed to vasoconstrictor peptides, inflammatory cytokines and physical factors (e.g. angiotensin II, thrombin, TGF-β). Although the human kidney contains mRNA for all three isoforms of endothelin (ET), ET-1 appears to be the only peptide expressed at the protein level [4]. Expression of ET-1, its precursor, big ET-1, and the ECE in the non-diseased kidney is largely confined to the glomerular and vascular endothelium [4, 5]. However, when proteinuria is present, there is tubular expression of ET-1 as well [6]. ET-1 released by the renal vasculature or nephron segments mainly acts on cells in the immediate vicinity in an autocrine/paracrine fashion. ET-1 mediates its biological effects by interacting with two receptors, the ETA and ETB receptors, which have been cloned and well characterized. The ETA receptor is preferentially expressed in vascular smooth muscle cells and mediates the potent constrictor actions of ET-1 [7]. The ETB receptor is primarily expressed in endothelial cells and binds all three ET isoforms. When this receptor is activated, nitric oxide and prostacyclin are released, possibly as a feedback loop to the constrictor actions of ET-1. This is exemplified by recent observations where both administration of a selective ETB antagonist in animals as well as specific knock-out … * Corresponding author. Tel: +31-30-250-7329; Fax: +31-30-254-3492; E-mail: t.rabelink@digd.azu.nl

Human endothelial cell storage granules: a novel intracellular site for isoforms of the endothelin-converting enzyme.

We have previously shown endothelin (ET)-like immunoreactive staining in Weibel-Palade bodies, storage granules that are an integral component of the regulated secretory pathway in endothelial cells. These structures degranulate after chemical or mechanical stimuli that result in cytosolic calcium influx. We therefore investigated whether the regulated pathway might be an intracellular site involved in the cleavage of big ET-1 to the biologically active peptide ET-1 by determining the ultrastructural localization of endothelin-converting enzyme (ECE)-1. A low level of ECE-like immunoreactivity was detected on the cell surface of human umbilical vein and coronary artery endothelial cells by scanning electron microscopy. Exogenous big ET-1 was added to permeabilized and nonpermeabilized cultured human umbilical vein endothelial cells, and ECE activity was measured by the detection of ET-like immunoreactivity in the culture supernatant. A marked increase in ECE activity was observed in permeabilized cells, indicating that ECE may also be expressed in intracellular compartments. Confocal microscopy revealed intense immunofluorescence staining for big ET-1 and the 2 isoforms of ECE-1 (ECE-1alpha and ECE-1beta) in the perinuclear region and in Weibel-Palade bodies of the human umbilical vein endothelial cells. Stimulated degranulation of storage granules by the calcium ionophore A23187 caused release of ET into the culture supernatants. The findings of this study indicate that big ET-1 is processed to the mature vasoactive peptide by ECEs located within endothelial storage granules. We hypothesize that this activity may be important in the regulated mobilization of ET in human endothelial cells.

Purification of a novel endothelin-converting enzyme specific for big endothelin-3

Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a K m value of 0.14 μM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET B receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.

Contribution of endothelin to the acute pressor response of L-NAME in stroke-prone spontaneously hypertensive rats.

In this study, we examined whether endothelin (ET) plays a role in the short-term increase in mean arterial pressure (MAP) after nitric oxide synthase (NOS) inhibition with N(omega)-nitro-L-arginine methyl ester (L-NAME) in stroke-prone spontaneously hypertensive rats (SHRSPs). Experiments were performed by using Inactin-anesthetized male SHRSPs that were pretreated with chlorisondamine to block reflex autonomic cardiovascular effects. Injection of L-NAME (10 mg/kg, i.v.), but not D-NAME, produced rapid and marked increases (74 +/- 3 mm Hg) in MAP that were sustained for >1 h. In SHRSPs that were treated with the ET(A/B) receptor antagonist, L-754,142 (15 mg/kg + 15 mg/kg/h), L-NAME increased MAP by 45 +/- 4 mm Hg (p < 0.0001 compared with L-NAME alone). L-754,142 blocked pressor responses to big ET-1 by >90% but was without effect on pressor responses to norepinephrine. Plasma levels of ET-1 averaged 5 +/- 1 pg/ml in animals given vehicle and were slightly increased in animals given either L-NAME alone (7 +/- 2 pg/ml) or L-754,142 alone (7 +/- 2 pg/ml) but increased markedly when L-NAME and L-754,142 were given together (114 +/- 18 pg/ml). This may relate to an effect of L-754,142 to block ET-receptor-mediated clearance of ET-1. We conclude that ET plays a role in the short-term pressor response after NOS inhibition in SHRSPs.

Immunocytochemical studies on endothelin in mast cells and macrophages in the rat gastrointestinal tract.

To reveal the distribution of endothelin (ET)-containing stromal cells (mast cells and macrophages), we investigated the rat gastrointestinal tract immunohistochemically using antibodies to Big ET-1, Big ET-2, Big ET-3, and mature ETs. In all the regions of the gastrointestinal tract, immunoreactivity for all the antibodies used was found in stromal cells that were located mainly in the lamina propria (not in the submucosa). The number of these cells was largest in the small intestine and smallest in the colon. Moreover, Big ET-2, which was originally identified in the gastrointestinal tract, was also found in many stromal cells, but Big ET-3-containing cells, unexpectedly, were found in almost the same number as Big ET-2-containing cells, while Big ET-1-containing cells were few. These immunopositive stromal cells seemed to be mast cells and macrophages from their histological features. Double-immunohistochemical staining revealed that 92% of the mature ETs-positive cells were mast cells; the rest were macrophages. Furthermore, we confirmed that mature ETs coexisted with ET-A or ET-B receptors in identical cells. Hence, we presume that ETs are synthesized in and secreted from stromal cells in the rat gastrointestinal tract, that their main isotypes are not only ET-2 but also ET-3, and that ETs may act in an autocrine/paracrine fashion.

Regional variability in preproEndothelin-1 gene expression in sheep pulmonary artery and lung during the onset of air-induced chronic pulmonary hypertension. Participation Of arterial smooth muscle cells.

We investigated preproendothelin-1 (ppET-1) gene expression in the main and midregion pulmonary artery, and peripheral lung from control sheep and from animals during the development of the structural and functional changes of air-induced chronic pulmonary hypertension (CPH). Measurement of ET-1 in lung lymph (n = 7) at 1, 4, 8, and 12 d of continuous air embolization (CAE) showed a significant increase from day 4 compared with controls (n = 4). A semiquantitative reverse transcription PCR for ppET-1 gene expression was developed using ovine-specific primers. Control sheep showed strikingly fewer ppET-1 transcripts in the midregion (22.9+/-2.3 ng cDNA equivalents) than in the main pulmonary artery and lung (736.0+/-263.7 and 705.5+/-125.7, respectively). Smooth muscle cells (SMC) isolated from the main and midregion artery of control sheep confirmed these findings and showed higher levels of intracellular ET-1 synthesis in the main versus the midregion artery. Differences in gene expression persisted during CAE. In main pulmonary artery and lung, ppET-1 transcripts fell to < 1% of controls. However, transcripts in the midregion artery showed a gradual increase. Coincubation of SMC from the midregion with ET-1 plus TGF-beta resulted in an increase in intracellular big ET-1 and a decrease in SMC from the main artery. We conclude that SMC from the main and midregion pulmonary artery are phenotypically different and suggest that local synthesis of ET-1 and TGF-beta, and increased levels of ET-1 in lung lymph, regulate ppET-1 gene expression and synthesis in arterial SMC during the development of air-induced CPH.

In vitro enzymatic processing of radiolabelled big ET-1 in human kidney

We have investigated enzymatic processing of big ET-1 in sections of human renal cortex by examining selected binding characteristics of the radiolabelled precursor and cleaved peptide. Sections of histologically normal human kidney obtained from patients undergoing nephrectomy for hypernephroma (50–74 years, N = 10, male or female) were incubated with 0.1 nM [ 125I]-ET-1, [ 125I]-Tyr 13 big ET-1 or [ 125I]-Tyr 31 big ET-1 in culture media at 37° to facilitate enzymatic activity. Specific binding measured from sections incubated with [ 125I]-Try 13 big ET-1 (which would yield [ 125I]-ET-1 on enzymatic cleavage) was 39.7 ± 2.5%. This was significantly reduced to 19.0 ± 2.0% following co-incubation with 10 μM thiorphan, an inhibitor of neutral endopeptidase (NEP) but not the putative endothelin converting enzymes (ECE). No further reduction in specific binding was obtained with 100 μM thiorphan, indicating that this is a maximal effect. However phosphoramidon (100 μM), an inhibitor of ECE and NEP, almost abolished specific binding, indicating that both NEP and ECE cleave big ET-1 in the kidney. No specific binding was detected when sections were labelled with [ 125I]-Tyr 31 big ET-1 (which would be expected to yield [ 125I] labelled C-terminal fragment). Binding of the product of processed [ 125I]-Tyr 13 big ET-1 was inhibited mainly by the ET B selective antagonist ( BQ788 = 75.1 ± 2.1% inhibition; FR139317 = 9.7 ± 7.3% inhibition), consistent with the predominance of this subtype in human kidney. We conclude that big ET-1 is processed by NEP and ECE in human kidney and that the cleaved product binds predominantly to the ET B receptor subtype. ECE may be a therapeutic target in the attenuation of renal diseases in which ET-1 has been implicated.

Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme-1 gene

Recent gene targeting studies have revealed unexpected roles for endothelins in the development of neural crest-derived tissues. Endothelin converting enzyme-1 (ECE-1) catalyzes the proteolytic activation of big endothelin-1 to endothelin-1(ET-1) in vitro. However, the importance of ECE-1 cleavage in the multiple endothelin pathways in vivo is unknown. Here we generated a targeted null mutation in the mouse ECE-1 gene. ECE-1-/- term embryos exhibited craniofacial and cardiac abnormalities virtually identical to the defects seen in ET-1 and endothelin A receptor (ETA)-deficient embryos. Epidermal melanocytes as well as enteric neurons of the distal gut were also absent in ECE-1-/- embryos, reproducing the developmental phenotype seen in ET-3-/- and endothelin B receptor (ETB)-/- mice. Surprisingly, large amounts of mature ET-1 peptide are found in ECE-1-/- embryos, indicating that non-ECE-1 protease(s) can activate ET-1 at certain sites. However, these enzymes cannot produce sufficient mature endothelin at the locations crucial for normal embryonic development. These findings reveal that ECE-1 is a bona fide activating protease for both big ET-1 and big ET-3 in vivo, and that the cell-cell communication pathways represented by the ET-1/ECE-1/ETA axis and the ET-3/ECE-1/ETB axis are each involved in the development of distinct subsets of neural crest cell lineages. Mutations in ECE-1 may cause developmental defects in humans, such as Hirschsprung disease, velocardiofacial syndrome and related neurocristopathies.

Novel Selective Quinazoline Inhibitors of Endothelin Converting Enzyme-1

PD 069185 is a highly selective and structurally novel inhibitor of endothelin converting enzyme-1 (ECE-1). PD 069185 is a trisubstituted quinazoline with an IC50value of 0.9 ± 0.1 μM for inhibition of human ECE-1 from the solubilized membrane fraction of CHO cells stably transfected with human ECE-1 cDNA. Kinetic analysis revealed that PD 069185 is best fit with a competitive inhibition model with a Kivalue of 1.1 ± 0.1 μM and binds in a reversible manner. The closely related enzyme, ECE-2, is not inhibited at up to 100 μM PD 069185. In addition, PD 069185 at 200–300 μM has little effect on other metalloproteases, such as neutral endopeptidase 24.11, stromelysin, gelatinase A, and collagenase, showing a high ECE-1 specificity. Data are also presented to show that this series of inhibitors are effective in inhibiting ECE-1 in intact cells and in attenuating the increase in perfusion pressure induced by big ET-1 in isolated rat mesentery. These non-peptidic ECE-1 inhibitors should serve as a valuable tool to study the pathophysiological role of endothelin and the therapeutic potential of ECE-1 inhibitors.

Big endothelin analogues with inhibitory activity on endothelin-converting enzyme-1.

Big endothelin-1 (big ET-1) is converted into an active form, ET-1, by endothelin-converting enzyme-1 (ECE-1). To assess the mechanism of substrate recognition by ECE-1, we examined the effects of variously substituted analogues of big ET-1 on ECE-1 activity, using solubilized membranes prepared from human ECE-1-expressed CHO-K1 cells. Among the big ET-1 analogues tested, [21Phe]big ET-1[18-34] and [31Ala]big ET-1[18-34] exhibited significant inhibition of ECE-1. A kinetic analysis revealed [21Phe]big ET-1[18-34] to be a competitive inhibitor (Ki = 21 microM) and [31Ala]big ET-1[18-34] to be a noncompetitive inhibitor (Ki = 36 microM). These results suggested that ECE-1 recognizes big ET-1 at the P1 position and the C-terminal region in a different manner and that modification of these regions can produce ECE-1 inhibitors.

Histochemical studies on endothelin and the endothelin-A receptor in the hypothalamus.

By use of a rabbit polyclonal antibody specific to the endothelin-A (ETA) receptor, we analyzed the distribution of neurons containing ETA receptors in rat brain. Almost all A1-A7 noradrenergic neurons, A8-A16 and retinal amacrine (A17) dopaminergic neurons (except the A15 group), and C3 adrenergic neurons contained ETA receptors. In addition, hypothalamic magnocellular (noncatecholaminergic) neurons also showed immunoreactivity to ETA receptors, whereas the staining intensity was low. These observations were confirmed by analyzing the expression of c-fos in the hypothalamus after central injection of ET isopeptides. We also examined immunohistochemically the distribution of big ETs and mature ETs in the hypothalamus. The paraventricular and supraoptic magnocellular neurons in the hypothalamus were immunopositive for big ET-1 and mature ET-1. However, their nerve fibers were immunostained only with antibody to big ET-1. The observations on big ET-1 were altered by colchicine treatment. Considering all the evidence, mature ET-1 may regulate neurotransmission in the hypothalamic region, whereas big ET-1 is secreted from nerve terminals into the vessels as a neurohormone. It is also concluded that ET, one of the "brain-vascular peptides," has a close relationship with the catecholamine neuron system in the brain.

Contractile activity of endothelins and their precursors in human umbilical artery and vein: identification of distinct endothelin-converting enzyme activities.

A number of studies using endothelin (ET) precursors, commonly termed big ETs, have revealed the presence of endothelin-converting enzyme (ECE) activity in various vascular and nonvascular preparations. Since then, more than one ECE has been cloned. It has also been observed that big ET-1 and big ET-3 are not converted by the same enzyme. The ECE responsible for big ET-3 conversion is rarely present because big ET-3 does not induce a contractile response in most isolated preparations tested. In this study we characterized ECE activities present in two human preparations, the umbilical artery and vein, testing the contractile activities of the three human Big ETs in the presence or absence of phosphoramidon, a dual ECE/neutral endopeptidase inhibitor. The results show that human big ET-1(1-38) is 6.3-fold more potent than big ET-2(1-38) in the human umbilical artery (an ETA preparation), whereas big ET-1 is equipotent to big ET-2 in the vein (which contains ETA and ETB receptors). Human big ET-3(1-41) is inactive on both vessels. Furthermore, phosphoramidon attenuated human big ET-1-induced contractions only in the umbilical artery and not in the vein. Such observations, in terms of substrate selectivity and phosphoramidon sensitivity, suggest the presence of distinct ECE activities in human vein and arteries.

Effect of the endothelin family of peptides on human coronary artery smooth-muscle cell migration.

The migration of coronary artery medial smooth-muscle cells (SMCs) is one of the key events in the process of intimal thickening in coronary atherosclerotic lesions. The objectives of the present study were to determine whether any of the three isoforms of endothelin (ET), ET-1, ET-2, and ET-3, or an intermediate form of ET, big ET-1, induces migration of human coronary artery SMCs, and to investigate the possible interaction of ET peptides and well-known migration-stimulatory factors, platelet-derived growth factor (PDGF)-BB and angiotensin II (Ang II), on SMC migration by the Boyden's chamber method. None of the ET peptides alone induced SMC migration between 10(-9) and 10(-7) mol/L. In contrast, ET-1 and ET-2 significantly induced SMC migration in the presence of low concentrations of PDGF-BB (0.5 ng/mL) or Ang II (10(-9) mol/L), although ET-3 was less active (ET-1 = ET-2 > ET-3). In contrast, big ET-1 was without significant activity on PDGF-BB-or Ang II-induced SMC migration. The potentiation of SMC migration by ET peptides was clearly inhibited by the ETA receptor antagonist BG-123 in a concentration-dependent manner. These results suggest that the ET family of peptides, especially ET-1 and ET-2, can induce human coronary artery SMC migration in combination with PDGF-BB or Ang II, probably via ETA receptors. Taken together with the finding that the concentrations of ET, PDGF-BB and Ang II are locally increased at sites of endothelial injury, this indicates that ET may be an initial stimulus for human coronary artery medial SMC recruitment during coronary atherosclerosis, possibly in combination with PDGF-BB or Ang II.

Benzofused macrocyclic lactams as triple inhibitors of endothelin-converting enzyme, neutral endopeptidase 24.11, and angiotensin-converting enzyme.

The purpose of this study was to identify endothelin-converting enzyme (ECE) inhibitors that also possess inhibitory activity for neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). The ortho-substituted benzofused macrocyclic lactams, such as CGS 26670, are generally potent NEP inhibitors but poor ACE inhibitors. CGS 26670 inhibited ECE activity with an IC50 of 600 nM, whereas it inhibited NEP and ACE activities with IC50 values of 0.9 and > 10,000 nM, respectively. This compound also prevented the conversion of big endothelin-1 (big ET-1) to ET-1 by denuded porcine coronary arterial smooth muscle with an IC50 of 200 nM. The ACE inhibitory activity is greatly is greatly improved in metasubstituted benzofused macrocyclic lactams. For example, CGS 26582 inhibited ECE, NEP, and ACE activities with IC50 values of 620, 4, and 175 nM, respectively. When injected at 30 mg/kg i.v. in conscious rats, followed by a challenge with big ET-1 at 1 nmol/kg i.v., this compound suppressed by 44% the increase in mean arterial blood pressure owing to the generation of ET-1 by ECE. Because ECE, NEP, and ACE play regulatory roles in cardiovascular and renal function, triple inhibitors of these enzymes may represent a novel class of agents for treatment of cardiovascular and renal diseases.

Characterization of site-directed antisera against endothelin-converting enzymes.

Site-directed antisera have been developed against the two endothelin-converting enzyme-1 (ECE-1) isoforms cloned to date in humans, ECE-1 alpha and ECE-1 beta. Antisera were raised in rabbits against synthetic peptides corresponding to the deduced amino acid sequences that differ between ECE-1 alpha and ECE-1 beta. Antisera were highly selective for their corresponding antigen (titer 1 x 10(4)) and did not detect ET-1 or big ET-1. Furthermore, no detectable crossreactivity was observed between the different site-specific antisera and the other immunizing peptides, suggesting that the antisera would be selective for ECE-1 alpha and ECE-1 beta. Standard displacement curves have been developed to determine the levels of immunoreactive ECE-1 alpha and ECE-1 beta in solubilized microsomal fractions of human tissue. In conclusion, we have described the first production and characterization of site-directed antisera raised against ECE-1 alpha and ECE-1 beta capable of discriminating between the two ECE-1 isoforms. Furthermore, using these antisera, we have found that ECE-1 alpha appears to be the predominant isoform in human tissue.

Phosphoramidon-sensitive and -insensitive endothelin-converting enzyme in human megakaryoblastic cell lines.

The human megakaryoblastic cell lines HEL, MEG-01, and DAMI express preproendothelin-1 mRNA. This investigation was designed to find out whether they could also express endothelin-converting enzyme (ECE) and release mature endothelin (ET). RT-PCR applied to RNA isolated from the cell lines amplified fragments of the expected size. The amplified cDNA of MEG-01 was submitted to restriction enzymes, which generated the expected subfragments. Membrane ECE activity was phosphoramidon-sensitive, in contrast to the cytosolic activity capable of producing ET-1 from big ET-1. The three cell lines produced ir-ET in a time-dependent manner. These results show that human megakaryoblastic cell lines express functional, phosphoramidon-sensitive and insensitive ECE activity and produce mature ET.

Changes in plasma and amniotic fluid endothelin levels during pregnancy: Facts or artefacts?

Objective: To describe the evolution of immunoreactive endothelins (irETs) in maternal peripheral plasma and in amniotic fluid at different stages of pregnancy using two different radioimmunoassays. Study design: Peripheral blood samples were obtained from ten non-pregnant and eighty-four pregnant patients at different stages of pregnancy, but not in labor. Amniotic fluid samples were obtained in ten patients during the second trimester of pregnancy and in twenty-two patients at term. Endothelin concentrations were assayed using two different kits: by antigenic cross-reactions, the RPA 545 assay allowed the detection of ET-1, ET-2 and big ET-1; the RPA 555 assay allowed the detection of ET-1, ET-2 and ET-3. Results: Using the RPA 555 kit, no differences were observed in irET plasma levels between non-pregnant and pregnant patients whatever the gestational age. With the RPA 545 kit, the irET levels were significantly lower in pregnant patients during the first and second trimesters of pregnancy when compared to non-pregnant patients. Immunoreactive ET levels then increased significantly in the last month of pregnancy when compared to mid-pregnancy levels. In amniotic fluid, irET levels were significantly higher at term than during the early second trimester, without any difference between the two RIA kits. Conclusion: Our results are indicative of a differential evolution in ET isoforms during pregnancy in maternal peripheral blood. The increase in irET observed towards the end of pregnancy in maternal plasma and in amniotic fluid suggests that ET could play a role in the onset of parturition.

Endothelin-converting enzyme in the human vasculature: evidence for differential conversion of big endothelin-3 by endothelial and smooth-muscle cells.

Our aim was to localize endothelin-converting enzyme (ECE) in human saphenous vein grafts and to quantify enzymic activity in cultured human endothelial and smooth-muscle cells. Immunoreactive ECE localized to the endothelium and infiltrating macrophages in vein grafts, but little or no immunoreactivity was detected within the media or proliferated smooth muscle of the occlusive lesion. Cultures of human umbilical vein endothelial cells were incubated with big endothelin-1 (ET-1) (10 nM) to measure extracellular conversion. After 2 h the concentration of mature peptide in the medium was increased by 162.7 +/- 21.6 pM (n = 3 +/- SEM) above basal. Permeabilization of the cells increased conversion to 1077.9 +/- 52.8 pM, suggesting that about 85% of ECE activity was located intracellularly. In both cases, activity was inhibited by phosphoramidon but not by thiorphan. In contrast, conversion of big ET-3 (10 nM) under the same conditions was not detected in either intact or permeabilized cells after 2 h. Big ET-3 and big ET-1 were converted by a phosphoramidon-sensitive/thiorphan-insensitive enzyme on the surface of confluent cultures of human umbilical vein smooth-muscle cells, with concentrations of the corresponding mature peptides increasing by 99.5 +/- 14.5 pM and 222.2 +/- 11.6 pM, respectively. These results suggest that smooth-muscle cells could be responsible for the synthesis of ET-3 present in plasma and for additional processing of big ET-1 released by endothelial cells.