Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We found that immunoreactive endothelin (ET) is present in seminal fluid in very large amounts (500-5000 ng/L; quantification based on ET-1 standard). This immunoreactive ET was detected by use of a radioimmunoassay system in which the N-terminal portion of ET-1 and ET-2 (and big ET-1 and big ET-2) are recognized. Thus, the immunoreactive ET in seminal fluid may include the precursors of ET-1 or ET-2 (i.e., big ET) as well as metabolites of ET-1 or ET-2 in which the N-terminal region is intact. The levels of immunoreactive ET in seminal fluid from men with normal semen analyses and that in seminal fluid of vasectomized men were within the same range. Using a different radioimmunoassay system in which the C-terminal portion of ET-1, ET-2, and ET-3 is recognized, we found that the levels of immunoreactive ET were much lower (or undetectable). We speculate that bioactive ET may be produced and act to promote sperm transport in the male reproductive tract; thereafter, bioactive ET may be metabolized by membrane metalloendopeptidase (which is present in male reproductive tissues and semen) to immunoreactive, inactive products. Alternatively, big ET in seminal fluid may be processed in tissues of the female internal genitalia to bioactive ET, which could act to promote sperm transport through the uterine cavity by stimulating myometrial contractions.
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PMID:Massive amounts of immunoreactive endothelin in human seminal fluid. 172 24

We have investigated the possible presence of endothelin-metabolizing neutral endopeptidase (NEP, EC 3.4.24.11) on A10 cell membranes using [125I]-ET-1 binding and direct measurements of NEP. NEP activity of A10 cell membranes has been compared to that of solubilized rat kidney brush border membranes (KNEP). Specific [125I]-ET-1 (50 pM) binding (defined with 100 nM ET-1) to A10 cell membranes was increased in a concentration dependent manner by the selective NEP inhibitors thiorphan, phosphoramidon, and SQ 28,603 [(+/-)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-beta-alanine] with EC50 values of 9.4, 28.4, and 5.7 nM respectively. At equilibrium (150 min), 70% more specific binding was apparent in the presence of these inhibitors. Phosphoramidon (2 microM) did not alter Bmax values, but it decreased the apparent KD for [125I] ET-1 from 63 (+/- 3) to 27 (+/- 2) pM. Thiorphan, phosphoramidon, and SQ 28,603 inhibited A10 cell NEP activity with IC50 values of 5.3, 36.5, and 6.0 nM respectively, which was similar to values obtained with KNEP (3.6, 22.6, and 3.5 nM). ET-1 inhibited A10 cell NEP, and KNEP with IC50 values of 30 and 21.3 microM respectively. The order of inhibitory potencies: ET-3 greater than ET-1 = ET-2 greater than or equal to sarafotoxin-6b was similar for both systems. These data suggest A10 cell membranes contain a NEP which has similar characteristics to NEP 24.11, and which actively metabolizes [125I]-ET-1.
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PMID:Vascular A10 cell membranes contain an endothelin metabolizing neutral endopeptidase. 201 30

A single addition of 3 x I0-7 M ET-1, ET-2 or ET-3 produced contractions that reached a steady state in 28.2 +/- 4.2, 21.1 +/- 1.3 and 24.0 +/- 3.8 min, respectively and took 2.7 +/- 0.4, 2.1 + 0.1 and 1.6 +/- 0.1 min to reach half of this steady-state response.4. Contractions induced by 3 x I0-7 M big ET-11-38 or big ET-11- 39 reached a plateau in 38.5 +/- 3.6 and 35.6 +/- 3.3 min, respectively, and half of these responses were attained in 12.0 +/- 2.5 and 7.1 +/- 1.1 min.Thus, these contractions developed more slowly than those induced by ET-1. Contractions induced by 3 x 10-7 M big ET-21-38 were also much slower to develop than those to ET-2, for these took 49 +/- 2 min to reach plateau and 19.4 +/- 2.1 min to attain half that response. Contractions induced by 3 x 10-7 M big ET-31-41 amide took 50.2 +/- 3.7 min to reach a plateau and 27.3 +/- 3.0 min to reach half of this response.5. Phosphoramidon (0.1, 1 and 3 x 10-4 M) inhibited contractions induced by big ET-11.39. For instance,the contractions induced by 3 x 10-7 M big ET-11-39 were inhibited by 10-4 M or 3 x 10-4 M of phosphoramidon by 62.8 +/- 6.7% or 74.5 +/- 4.6%, respectively. Similarly, contractions induced by ET-21-38 were inhibited by 91.3 +/- 5.4% and the small response induced by big ET-3l-4l amide was abolished by 3 x 10-4M phosphoramidon. Conversely, the neutral endopeptidase (EC 24.11) inhibitor DL-thiorphan(3 x 10-4 M) had no effect. Captopril (10-5 M), pepstatin A (10-5 M), phenylmethylsulphonylfluoride(PMSF, 10-3 M), aprotinin (10-5 M), E-64 (10-5 M), cystatin (10-6 M), leupeptin (10-4 M),chymostatin (10-4 M), or bestatin (10-5 M) did not inhibit but rather increased to a similar, but small degree the contractions induced by 3 to 30 x 10-9 M big ET-11-39. Only captopril (10-5 M) or leupeptin(10-4 M) increased the contraction induced by 3 x 10-7 M big ET-11-39. Phosphoramidon (10-4 M),pepstatin (10-5 M) or PMSF (10-3 M) did not affect contractions induced by ET-1.6. Removal of the epithelium increased by 70% the size of the contraction induced by 5 microM histamine(1.08 +/- 0.05 g; n = 160 to 1.84 +/- 0.14 g; n = 12) but did not affect, in absolute terms, the contraction induced by ET-1 (as a % of the response to histamine, these responses were, of course, apparently depressed). Epithelium removal did, however, increase the size of the contractions induced by 3 to 30 x 10-9 M big ET-1 -39 which was very similar to the effect of the protease inhibitors.7. In competition binding studies on membranes prepared from the guinea-pig gallbladder, 10-11 MET-1 inhibited by 76.9 +/- 3.1% the binding of [125]-ET-I while porcine big ET-11-39 caused no inhibition(0.7 +/- 3.0; n = 3). ET-1 (10-6 M) inhibited binding by 95.7 =/- 1.1% (n = 3) while at this much higher concentration, big ET-11-39 inhibited binding by only 16.8 +/- 4.2% (n = 3). This clearly suggests that big ET-11-39 does not bind directly to ET receptors.8. Thus, a phosphoramidon-sensitive endothelin-converting enzyme (ECE), different from neutral endopeptidase (NEP; EC 24.11) and not located on the epithelium, converts big ET-1 into ET-1 in the gallbladder of the guinea-pig. This ECE appears to act preferentially on big ET-1 or big ET-2 over bigET-3.
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PMID:Contractile activity of endothelin precursors in the isolated gallbladder of the guinea-pig: presence of an endothelin-converting enzyme. 760 42

Purification of endothelin converting enzyme (ECE) from endothelial cells has been hindered by the difficulty in obtaining primary endothelial cells in large quantity. We therefore tested transformed human umbilical vein endothelial cells (EA.hy926) for ECE activity. Our data clearly demonstrate that this transformed cell line preserves the ECE properties of the primary cell line. These include: (i) one sharp activity optimum at neutral pH; (ii) characteristics typical of a metalloprotease; (iii) IC50 value for phosphoramidon of 1.8 microM (2.7 microM for HUVEC); (iv) no inhibition by captopril and thiorphan, inhibitors of angiotensin converting enzyme and neutral endopeptidase 24.11. The enzyme showed a substrate specificity for big ET-1:big ET-2:big ET-3 in a ratio of 40:2.5:1. This report presents evidence that a permanent human endothelial cell line, EA.hy926, preserves the ECE activity of HUVEC and is useful for the study of ECE and its regulation of ET-1 production.
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PMID:A permanent human cell line (EA.hy926) preserves the characteristics of endothelin converting enzyme from primary human umbilical vein endothelial cells. 779 20

Endothelins (ET) are a family of potent vasoactive peptides that are produced from biologically inactive intermediates, termed big endothelins, via a proteolytic processing at Trp21-Val/Ile22. We recently cloned and characterized a membrane-bound metalloprotease that catalyzes this proteolytic activation, endothelin-converting enzyme-1 (ECE-1) (Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yanagisawa, M. (1994) Cell 78, 473-485). This enzyme was shown to function in the secretory pathway as well as on the cell surface. Here we report molecular cloning of another novel enzyme, ECE-2, that produces mature ET-1 from big ET-1 both in vitro and in transfected cells. The cDNA sequence predicts that bovine ECE-2 is a metalloprotease structurally related to ECE-1, neutral endopeptidase 24.11, and human Kell blood group protein. The deduced amino acid sequence of ECE-2 is most similar to ECE-1, with an overall identity of 59%. ECE-2 resembles ECE-1 in that it is inhibited in vitro by phosphoramidon and FR901533 but not by thiorphan or captopril, and it converts big ET-1 more efficiently than big ET-2 or big ET-3. However, ECE-2 also exhibits the following striking differences from ECE-1. (i) The sensitivity of ECE-2 to phosphoramidon is 250-fold higher as compared with ECE-1, while FR901533 inhibits both enzymes at similar concentrations. (ii) ECE-2 has an acidic pH optimum at pH 5.5, which is in sharp contrast to the neutral pH optimum of ECE-1. ECE-2 has a narrow pH profile and is virtually inactive at neutral pH. Chinese hamster ovary (CHO) cells, which lack detectable levels of endogenous ECE activity, secrete mature ET-1 into the medium when doubly transfected with ECE-2 and prepro-ET-1 cDNAs. However, ECE-2-transfected CHO cells do not efficiently produce mature ET-1 when present with an exogenous source of big ET-1 through coculture with prepro-ET-1-transfected CHO cells. These findings suggest that ECE-2 acts as an intracellular enzyme responsible for the conversion of endogenously synthesized big ET-1 at the trans-Golgi network, where the vesicular fluid is acidified.
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PMID:Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum. 779 12

Endothelin (ET) is a 21-residue potent vasoconstrictive peptide produced by vascular endothelial cells and formed from its precursor, big endothelin (big ET), by endothelin-converting enzyme (ECE). Here we report the cloning and functional expression of a complementary DNA encoding a rat ECE from endothelial cells. Rat ECE is a highly glycosylated protein consisting of 10 possible N-linked glycosylation sites, a zinc-binding domain, and a single membrane-spanning region. It has structural and sequence homology to neutral endopeptidase and Kell blood group protein, a putative neutral endopeptidase. Monoclonal antibody risen against purified rat lung ECE recognized broad glycosylated bands in membrane fractions prepared from both rat lung and COS cells transfected with a rat ECE expression vector. Expressed ECE was inhibited by phosphoramidon, but not by thiorphan. It also cleaved big ET-1 efficiently but not big ET-2 or big ET-3. Northern blot analysis revealed that ECE messenger RNA is widely expressed in many tissues.
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PMID:Cloning and functional expression of endothelin-converting enzyme from rat endothelial cells. 803 69

Endothelin-1 (ET-1), a 21 amino-acid potent vasoconstrictor peptide, is produced from the biologically inactive intermediate big ET-1 via an endoproteolytic cleavage between Trp-21 and Val-22 by endothelin converting enzyme (ECE). cDNA sequence analysis predicts that the two other members of the endothelin family, ET-2 and ET-3, are also generated from the corresponding intermediates called big ET-2 and big ET-3, respectively. The metalloproteinase inhibitor phosphoramidon inhibited the conversion of big ET-1 into mature ET-1 both in vivo and in cultured endothelial cells, suggesting that ECE may be a neutral metalloproteinase. In this study, we solubilized and partially purified ECE from the membrane fraction of porcine lung. Using gel filtration chromatography, we separated two distinct ECE activities, designated M1 (apparent molecular mass approx. 300 kDa) and M2 (approx. 65 kDa). Optimum pH for the cleavage of big ET-1 by M1 and M2 was 7.0 and 7.5, respectively. M1 efficiently converted human big ET-1(1-38) to ET-1, but not human big ET-2(1-37) or human big ET-3(1-41)-amide. In contrast, M2 converted both big ET-1 and big ET-2, but not big ET-3. M1 was inhibited by phosphoramidon (IC50 approx. 1 microM) but not by thiorphan or bacitracin. In contrast, M2 was inhibited by much lower concentrations of phosphoramidon (IC50 approx. 0.3 nM), as well as by thiorphan and bacitracin. ECE activity in M1 was able to bind to a concanavalin A-agarose column and was eluted by alpha-methyl-D-glucoside, indicating that the ECE is glycosylated. From these results, M1 and M2 from the porcine lung membrane are similar to the candidate of ECE in endothelial cells and neutral endopeptidase in kidney (EC 3.4.24.11), respectively. Taken in conjunction with the previous finding that neither thiorphan nor bacitracin affected the conversion of endogenously synthesized big ET-1 in cultured endothelial cells, we conclude that physiologically relevant ECE found in the endothelial cells is more similar to M1 than to M2.
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PMID:Characterization of phosphoramidon-sensitive metalloproteinases with endothelin-converting enzyme activity in porcine lung membrane. 843 80

A critical processing step in endothelin biosynthesis is the conversion of the intermediate "big endothelin" to its biologically active product catalysed by endothelin converting enzyme (ECE). In this commentary we discuss critically the cellular location, structure, and activity of the isoforms of ECE. The current evidence supporting a metallopeptidase ECE as the physiological regulator of endothelin production is described. Its sensitivity to inhibition by the fungal metabolite phosphoramidon and subsequent cloning of the enzyme indicate it to be a type II integral membrane protein homologous with neural endopeptidase-24.11 (E-24.11), the major neuropeptide-degrading ectoenzyme in brain and other tissues. Unlike E-24.11, however, ECE exists as a disulphide-linked dimer of subunit M(r) 120-130 kDa and is not inhibited by other E-24.11 inhibitors such as thiorphan. Alternative splicing produces two forms of ECE with distinct N-terminal tails. These isoforms of ECE-1 show similar specificity converting big endothelin-1 (ET-1) to ET-1 but big ET-2 and big ET-3 are converted much less efficiently. This suggests that additional forms of ECE remain to be isolated. Immunocytochemical studies indicate a predominant cell-surface location for ECE-1, like E-24.11. This is consistent with the conversion of exogenous big ET-1 when administered in vivo and the inhibition of this event by phosphoramidon. However, mature ET-1 can be detected in intracellular vesicles in endothelial cells, suggesting that some processing occurs in the constitutive secretory pathway. This may be mediated by ECE-2, a recently cloned member of the E-24.11/ECE family which has an acidic pH optimum. Selective inhibitors of ECE may have therapeutic applications in cardiovascular and renal medicine.
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PMID:Molecular pharmacology of endothelin converting enzymes. 861 90

Increasing evidence suggests that endothelin, a potent vasoconstrictor, is implicated in cyclosporin A (CsA)-induced nephrotoxicity. Increased levels of urinary and circulating endothelin have been described in CsA-treated humans and animals. The exact mechanisms by which CsA induces these increases are still unknown, and no data indicate whether these elevated levels reflect increased synthesis or decreased clearance of endothelin. In the present study, we investigated the effects of CsA administration (50 mg/kg per day i.p. for 6 days) to rats on plasma and urinary levels of endothelin; expression of endothelin-1 (ET-1), ET-3, and endothelin-converting enzyme in renal tissue; clearance of infused 125I-ET-1; and degradation of 125I-ET-1 by recombinant neutral endopeptidase. Rats given CsA for 6 days developed severe renal insufficiency, as shown by a 74% decrease in creatinine clearance rate (Ccr) (P < .006). Ccr was remarkably improved in CsA-treated rats that received bosentan, the combined antagonist of both endothelin A and endothelin B receptors. Urinary excretion of endothelin increased from an undetectable level to 31.7 +/- 6.0 pg/24 h (P < .001), and plasma levels of endothelin were unchanged (2.8 +/- 0.2 to 3.1 +/- 0.2 pg/mL). Reverse transcription followed by quantitative polymerase chain reaction revealed that ET-1 mRNA in the renal medulla increased by 59% (P < .006), whereas the expression of both ET-3 and endothelin-converting enzyme was unchanged. In other rats, neither acute nor chronic treatment with CsA affected either the clearance of 125I-ET-1 from the blood or the renal and pulmonary uptake of the peptide. Moreover, CsA did not affect the degradation of 125I-ET-1 by highly purified recombinant neutral endopeptidase, a well-known endothelinase. Taken together, these data suggest that the elevated urinary endothelin levels obtained after CsA treatment originate from the kidney and reflect increased renal synthesis of ET-1. Moreover, the production of endothelin appears to be regulated at the mRNA transcription level, and expressions of ET-1 and ET-3 are regulated independently.
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PMID:Effects of cyclosporin A on the synthesis, excretion, and metabolism of endothelin in the rat. 862 Dec 8

The formation of endothelins (ET) from exogenous big-ET-1, -2 and -3 was investigated in cultured guinea pig Clara cells. When Clara cells were incubated with 10(-9) or 10(-8) M human big-ET-1 (1-38), the release of ET was 69.9 +/- 7.6 and 221.6 +/- 25 fmol/ml respectively, after 1 h incubation period. Treatment of Clara cells with 1 mM phosphoramidon strongly suppressed (80%) the conversion of big-ET-1 to ET-1 during 1, 2 and 6 h incubation periods. Treatment of Clara cells with thiorphan (1 mM), an inhibitor of neutral endopeptidase, decreased by 44 and 26% the levels of immunoreactive endothelin (ir-ET) in the cell supernatant respectively, after 1 and 2 h incubation periods. When Clara cells were incubated with 10(-8) M human big-ET-2 (1-38), the release of ir-ET was 287.6 +/- 8.9 fmol/ml after a 1 h incubation period. Phosphoramidon and thiorphan (1 mM) reduced the amount of ir-ET generated from exogenous human big-ET-2 (10(-8) M) by 69 and 56%, respectively. When Clara cells were incubated with 10(-8) M human big-ET-3 (1-41), the release of ET was not increased after a 1 h incubation period. Our results suggest that guinea pig Clara cells convert exogenous big-ET-1 and -2 but not big-ET-3 into ET-1 and ET-2, respectively, through metalloproteinases sensitive to both phosphoramidon and thiorphan.
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PMID:Phosphoramidon and thiorphan suppress the generation of endothelin (ET) from exogenous big-endothelin by guinea pig Clara cells. 911 Mar 81


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