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PubMed1992

Rhamnose moiety of phosphoramidon is not required for in vivo inhibition of endothelin converting enzyme.

Experiments were conducted to determine the importance of the carbohydrate moiety of phosphoramidon in the inhibition of the pressor response to big endothelin-1 in anesthetized rats. Big endothelin-1 produced a 42% increase in mean arterial pressure which was nearly abolished by co-infusion of phosphoramidon. Similarly, when an analog of phosphoramidon lacking the rhamnose group (phosphoryl-leu-trp-OH) was co-infused, a significant attenuation of the pressor response was observed. These findings indicate that the rhamnose moiety of phosphoramidon is not responsible for the distinguishing this compound as an inhibitor of the response to big endothelin-1 in the rat.

Animals↗
PubMed1992

Biochemical properties of endothelin converting enzyme in renal epithelial cell lines.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme (ECE) in non-vascular cells (renal epithelial cell lines, MDCK and LLC-PK1). ECEs derived from these epithelial cells were very similar to the endothelial ECE in the following biochemical properties: 1) The optimum pH was 7.0; 2) the Km value for big ET-1 was approximately 30 microM; 3) the enzyme was potently inhibited by EDTA, o-phenanthroline and phosphoramidon; and 4) the enzyme did not convert big ET-2 or big ET-3. These data suggest that phosphoramidon-sensitive ECE is involved in the processing of big ET-1 to ET-1 in the renal tubule.

Animals↗
PubMed1992

Role of mast cell chymase in the extracellular processing of big-endothelin-1 to endothelin-1 in the perfused rat lung.

Previous studies of endothelin-1 (ET) synthesis have shown that some cultured endothelial cells secrete an intermediate product, big-endothelin-1 (bigET), suggesting that the processing of secreted bigET to ET may be physiologically significant. In this study, two pertinent ET converting enzyme activities, mast cell chymase I (EC 3.4.21.39) and a phosphoramidon-sensitive, neutral metalloprotease, were identified in a rat lung particulate fraction. We perfused rat lungs with bigET and chymostatin or phosphoramidon to study the relevance of these two proteases to the processing of extracellular bigET in vivo. Addition of compound 48/80 (a compound which activates mast cells, causing degranulation and release of chymase) to the perfusion buffer greatly increased hydrolysis of exogenously added bigET to ET. ET formation was inhibited completely by 32 microM chymostatin, whereas inhibition by 50 microM phosphoramidon was incomplete and variable. Perfusate histamine levels were used to monitor the extent of mast cell degranulation, and inhibition of ET production by phosphoramidon was attributed to inhibition of degranulation, per se. There was a direct correlation between perfusate ET and histamine levels in both control and phosphoramidon-treated (but not chymostatin-treated) lungs. Our results suggest that chymase from lung mast cells is capable of physiologically relevant extracellular processing by bigET to ET in the perfused rat lung.

Animals↗
PubMed1992

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.

Adenocarcinoma↗
PubMed1992

Identification of endothelin converting enzyme in bovine lung membranes using a new fluorogenic substrate.

An enzyme partially purified from bovine lung membranes appears to be endothelin converting enzyme (ECE). This enzyme specifically cleaves big endothelin-1 (big ET-1) at the proper site, between Trp21 and Val22, with maximum activity at pH 7.5 and with a Km of roughly 3 microM, to produce endothelin-1 (ET-1) and C-terminal peptide (CTP). This same enzyme hydrolyzes the fluorogenic substrate succinyl-Ile-Ile-Trp-methylcoumarinamide to release the highly fluorescent 7-amino-4-methylcoumarin. The peptide derivative has the same amino acid sequence as big ET-1 and is a good substrate with a Km of about 27 microM. This enzyme is a metalloproteinase. It is not inhibited by five common proteinase inhibitors (pepstatin A, PMSF, NEM, E-64 and thiorphan) but it is inhibited by phosphoramidon and chelating compounds. The apoenzyme is restored to nearly full activity by a zinc-EDTA buffer with pZn = 13.

Animals↗
PubMed1992

A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.

We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.

Animals↗
PubMed1991

Importance of the C-terminal region of big endothelin-1 for specific conversion by phosphoramidon-sensitive endothelin converting enzyme.

Based on our previous findings that phosphoramidon-sensitive endothelin (ET) converting enzyme (ECE) converts human big ET-1 but does not big ET-3, we investigated structural requirement for substrate peptide. We prepared shorter peptides of big ET-1 and measured hydrolysis of the Trp-Val bond of these peptides. Relative hydrolysis ratios of big ET-1(1-38), (1-37), (16-37), (1-31) and (17-26) were 1, 1.15, 3.71, 0.01 and 0, respectively. In addition, big ET-2 and big ET-3 were not significantly converted by ECE. These results suggest that the carboxyl-terminal sequence at residues 32-37 of big ET-1 is important for conversion, whereas the amino-terminal disulfide loop structure appears to interfere with access of ECE to big ET-1.

Amino Acid Sequence↗
PubMed2025

Identification of ECE2 signaling in promoting non-small lung cancer progression through ET1/YAP1/MAGEA3 axis.

Non-small cell lung cancer (NSCLC) is a major cause of cancer-related mortality worldwide with high heterogeneity. However, the molecular basis for NSCLC development remains poorly understood. In this study, we analyzed endothelin converting enzyme 2 (ECE2) expression in NSCLC using transcriptome data from 59 normal and 515 NSCLC tissues obtained from The cancer genome atlas (TCGA) database. Additionally, we investigated the role of ECE2 in metastasis using 30 clinical NSCLC specimens. In vitro cell proliferation and migration assays were conducted using CCK8 and Transwell assays in NSCLC cells overexpressing ECE2. We employed Western blotting and immunostaining to assess activation of the endothelin-1 (ET1)/YAP1/MAGEA3 pathway. Furthermore, in vivo studies using subcutaneous xenograft mouse models with vector and ECE2-overexpressing A549 cells evaluated the anticancer effects. Our findings revealed elevated ECE2 expression in NSCLC tissues associated with poor prognosis. Moreover, overexpression of ECE2 enhanced both the proliferative and metastatic potential of NSCLC cells. Mechanistically, ECE2 promoted the production of ET1 in NSCLC cells. Subsequently, increased ET1 levels activated the YAP1/MAGEA3 pathway, thereby facilitating tumor progression. Our study uncovered the oncogenic role of ECE2 in promoting NSCLC growth through the ET1/YAP1/MAGEA3 pathway. Inhibiting ET1 signaling markedly enhanced the anticancer effectiveness of paclitaxel (PTX), providing a promising approach for managing NSCLC.

Humans↗
PubMed1991

Conversion of big endothelin isopeptides to mature endothelin isopeptides by cultured bovine endothelial cells.

Both the soluble and membrane fractions prepared from cultured bovine endothelial cells (ECs) possessed the converting activities to metabolize big endothelin-1 (big ET-1) to endothelin-1 (ET-1) at neutral pH. Metal chelators inhibited the activities of both fractions, whereas phosphoramidon, a metalloprotease inhibitor, strongly inhibited only the activity of the membrane fraction. Phosphoramidon reduced the secretion of ET-1 and concomitantly enhanced the release of big ET-1 from cultured ECs. The incubations of big ET-1, big ET-2, and big ET-3 with cultured ECs resulted in their conversions to mature ETs. Phosphoramidon also abolished these conversions. These results indicate that vascular endothelium can convert not only endogenous big ET-1 but also exogenous big ET isopeptides to their mature ETs through a phosphoramidon-sensitive neutral metalloprotease.

Animals↗
PubMed1993

Endothelin-2-converting enzyme from human renal adenocarcinoma cells is a phosphoramidon-sensitive, membrane-bound metalloprotease.

From the membrane fraction of cultured human renal adenocarcinoma (ACHN) cells, two endothelin-2-converting enzymes (ECE-2A and ECE-2B) were solubilized with detergent Lubrol PX and separated by hydrophobic butyl fast-performance liquid chromatography. The pH range of the converting activity of ECE-2B for big endothelin-1 (big ET-1), big ET-2, or big ET-3, was very narrow, and the optimal pH for each substrate was significantly different; the pH optimum for big ET-1 was 6.8 and that for big ET-2 or big ET-3 was 6.4. The ET-converting activity was abolished by phosphoramidon, 1,10-phenanthroline and EDTA but was not inhibited by thiorphan, E-64, leupeptin, PCMS, p-APMSF, or pepstatin A. The conversion efficiency for big ET-2 or big ET-3 by ECE-2B was approximately one-eighth of that for big ET-1. The molecular weight of ECE-2B was estimated to be 400 kDa by gel filtration. Because these characteristics of ECE-2B are very similar to those of ET-1-converting enzyme (ECE-1) in endothelial cells, these results raise the possibility that ECE-2B is identical to ECE-1 and that a single ECE physiologically converts all big ETs to the corresponding ETs in ET-producing cells.

Adenocarcinoma↗
PubMed1991

Different pharmacological profiles of big-endothelin-3 and big-endothelin-1 in vivo and in vitro.

1. Human big-endothelin-1 (big-ET-1) and endothelin-1 (ET-1) are equipotent as pressor agents and produce a significant change in mean arterial blood pressure (MAP) in anaesthetized guinea-pigs (2 nmol kg-1: peak delta MAP: 23 +/- 6 mmHg and 26 +/- 5 mmHg, respectively). 2. Unlike big-ET-1, big-endothelin-3 (big-ET-3) (10 and 20 nmol kg-1) induces no pressor responses whereas endothelin-3 (ET-3) at 2 nmol kg-1 induces a significant increase of blood pressure in anaesthetized guinea-pigs (peak delta MAP: 27 +/- 5 mmHg) with a shorter duration than ET-1 and big-ET-1. 3. Big-ET-1 at concentrations 40 times higher than those required for ET-1 (2.5 nM) releases prostacyclin (PGI2) (maximal release: 2.7 +/- 0.8 ng ml-1; 2.9 +/- 0.9 ng ml-1, respectively) and thromboxane B2 (TxB2) (maximal release: 6.7 +/- 1.3 ng ml-1; 6.8 +/- 1.1 ng ml-1, respectively) from guinea-pig perfused lungs. ET-3 (2.5 nM) is also a potent releaser of PGI2 and TxB2 from the guinea-pig lungs (maximal release: PGI2: 2.4 +/- 1.0 ng ml-1; TxB2: 3.8 +/- 0.6 ng ml-1). Conversely, big-ET-3 (100 nM) does not increase basal release of eicosanoids. 4. Phosphoramidon (50 microM), a metalloprotease inhibitor, markedly reduced the eicosanoid releasing properties of big-ET-1 (n = 4, P less than 0.01) in guinea-pig perfused lungs without affecting the release stimulated by ET-1. 5. Our results suggest that big-ET-1 is converted to ET-1 via a phosphoramidon-sensitive endothelin converting enzyme (ECE) to release eicosanoids. The ECE is present in the guinea-pig pulmonary vasculature. Furthermore, our results suggest that the ECE activity is specific for big-ET-1 and may not convert big-ET-3 to its active metabolite, ET-3.

Animals↗
PubMed1995

Effects of phosphoramidon on endothelin-1 and big endothelin-1 production in human aortic endothelial cells.

Using cultured human aortic endothelial cells, we examined the effects of phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, on the release of endogenous endothelin-1 (ET-1) and big endothelin-1 (big ET-1), and on the generation of ET-1 from exogenously applied big ET-1. Phosphoramidon, at concentrations of 10(-6) to 2 x 10(-4) M, caused a biphasic alteration of the ET-1 release, i.e., at lower concentrations of the drug, there were slight but unexpected increases of the release, whereas higher concentrations led to a decrease which is due to the drug-induced inhibition of ECE. The former effect appears to be based on the inhibition of ET-1 degradation by neutral endopeptidase 24.11 (NEP), since kelatorphan, a specific NEP inhibitor, produced a similar increasing effect on ET-1 release. Phosphoramidon enhanced the big ET-1 release from the cells in a concentration-dependent manner. When high concentrations of phosphoramidon were added, there was a dramatic increase in the release of big ET-1, which cannot be explained only by the drug-induced inhibition of ECE. This increase in big ET-1 release appeared to be partly due to a transient stimulation of the expression of prepro ET-1 mRNA. The amount of ET-1 generated from exogenously applied big ET-1 was markedly decreased by phosphoramidon in a concentration-dependent manner. In a similar fashion, phosphoramidon markedly inhibited ECE activity of the membrane fraction of cultured cells. Thus, ET-1 generation from exogenously applied big ET-1 reflects the functional phosphoramidon-sensitive ECE activities in human aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗
PubMed1995

WS75624 A and B, new endothelin converting enzyme inhibitors isolated from Saccharothrix sp. No. 75624. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

Novel endothelin converting enzyme (ECE) inhibitors, WS75624 A and B, have been isolated from the fermentation broth of Saccharothrix sp. No. 75624. These inhibitors were purified from an acetone extract of whole culture broth followed by HP-20 column chromatography, silica gel column chromatography and HPLC. WS75624 A and B showed highly potent ECE inhibitory activity, and both had IC50 values of 0.03 microgram/ml. WS75624 A and B also showed other metalloprotease (collagenase and neutral endopeptidase) inhibitory activity with IC50 values of 1 microgram/ml. Since large amount of WS75624 B was isolated, we tried in vivo evaluation using WS75624 B. WS75624 B inhibited big endothelin-induced pressor effect when administered to SD rat intravenously with big ET-1.

Animals↗
PubMed1991

Phosphoramidon-sensitive endothelin converting enzyme in rat lung.

A neutral proteinase with endothelin (ET)-1 converting activity was identified in cytosol and membrane fractions prepared from rat lung, in a ratio of 1:4, respectively. The membrane-bound proteinase was solubilized by 0.5% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) with an increase in specific activity, and then was characterized. The solubilized proteinase was capable of converting big ET-1 to ET-1 with an optimum pH of 6.5, and the conversion was dose-dependently suppressed by phosphoramidon (IC50 = 0.5 microM). The molecular mass of the proteinase was estimated to be about 500 kDa by gel filtration in the presence of 0.5% CHAPS. These results indicate that rat lung contains a phosphoramidon-sensitive neutral proteinase catalyzing conversion of big ET-1 to ET-1. The proteinase may be involved in the biosynthetic pathway of ET-1 in the lung and/or the conversion of circulating big ET-1.

Animals↗
PubMed1995

Characterization of endothelin receptors mediating mechanical responses to the endothelins in the isolated stomach strip of the rat.

We have characterized the receptors mediating the mechanical responses of the isolated stomach strip to endothelin-1 (ET-1), endothelin-3 (ET-3) and the ETB-selective receptor agonists sarafotoxin 6c (SX6c) and IRL 1620. As antagonists we used BQ-123 (ETA receptor selective), BQ-788 (ETB receptor selective) and PD 145065 (ETA/ETB receptor nonselective). We have also compared the responses of the mature peptides to their precursors human big ET-1(1-38), porcine big ET-1(1-39) and big ET-3(1-41) amide. ET-1, ET-3, SX6c and IRL 1620 produced equipotent concentration-dependent contractions of the rat stomach strips that were antagonized by PD 145065 (10(-5) M) or BQ-788 (10(-5) M) but not by BQ-123 (10-5 M). This indicates that the ETB receptor mediates contractions to the endothelins in this preparation. In preparations precontracted with PGE2 (3 x 10(-8) M), ET-1, but not SX6c (both 3 x 10(-9) M), caused a transient (< 2 min) relaxation (approx. 40% of the induced tone). This relaxation was antagonized by BQ-123 (10(-5) M) but prolonged by BQ-788, and therefore mediated by ETA receptors. A single administration of 3 x 10(-7) M ET-1, ET-3, SX6c or IRL 1620 produced contractions that reached a maximal response after 1 to 3 min. The contractions were not maintained, although responses to ET-1 or ET-3 lost their tone less rapidly than those to SX6c or IRL 1620.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗