Regulation of endothelin-converting enzyme-1 expression in human neuroblastoma cells

In recent years endothelin-converting enzyme (ECE-1) has been suggested to play an important role in amyloid-beta peptide metabolism as one of the amyloid-degrading enzymes. In this connection, the analysis of the levels of expression and distribution of ECE-1 in the brain under normal and pathologic conditions could be important in neurodegeneration and pathogenesis of Alzheimer disease. In our previous studies, we have demonstrated that expression of ECE-1 was significantly reduced in the cortex of adult rats after 15 mins of global ischemia. It was also significantly reduced in the striatum of rats subjected to prenatal hypoxia. In the present study, we analyzed effects of hypoxia and oxidative stress on ECE-1 in human neuroblastoma NB7 cells and effects of the cholinergic agonist carbachol and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). We have found that chronic (24 hrs) hypoxia and oxidative stress resulted in 30% and 20% decrease in expression of ECE-1 at the protein level, respectively, although at the level of ECE-1 mRNA there were no statistically significant changes. Serum withdrawal from the incubation medium as well as addition of carbachol or PMA for 24 hrs also led to a significant reduction of the levels of ECE-1 protein in NB7 cells. Further study of the downstream signaling cascades involved in downregulation of ECE expression in NB7 cells and primary neuronal cells might provide us with new insights into possible therapeutic strategies for prevention or treatment of Alzheimer disease in elderly patients and those who suffer from stroke or cerebrovascular disorders.     

Hydrogen peroxide regulation of bovine endothelin-converting enzyme-1

Vascular injury leads to the production of reactive oxygen species (ROS), but the mechanisms by which ROS contribute to vascular pathology are not completely understood. We hypothesized that ROS increase endothelin converting enzyme (ECE-1) expression. We found that glucose oxidase (GO) increases ECE-1 mRNA, protein, and activity in bovine aortic endothelial cells. Catalase abolishes this effect. Glucose oxidase treatment of endothelial cells transactivates the ECE-1 promoter. The ECE-1 promoter element that mediates this response to GO is located between -444 and -216 bp. This region contains a STAT response element, and GO activates STAT-3 binding to this STAT response element. Our data suggest that STAT3 mediates hydrogen peroxide induction of ECE-1 expression.     

Native and oxidized low-density lipoproteins stimulate endothelin-converting enzyme-1 expression in human endothelial cells

This study addressed the question how different lipoproteins modulate the expression of endothelin-converting enzyme-1 (ECE-1) in human endothelial cells. The effect of native and oxidized low-density lipoproteins (nLDL, oxLDL) on expression of ECE-1, prepro-endothelin-1, and endothelin-1 peptide was studied in primary cultures of human endothelial cells. Native and oxidized LDL increased ECE-1 mRNA after 1 h, reaching its maximum at 100 microg/ml (1.9- and 2.5-fold, respectively). Furthermore, ECE-1 protein expression, prepro-endothelin-1 mRNA, and endothelin-1 peptide release were increased in response to nLDL or oxLDL. Induction of ECE-1 by nLDL and of prepro-endothelin-1 by oxLDL was reduced by protein kinase C inhibition. Increased expression of ECE-1 mRNA by oxLDL and of prepro-endothelin-1 by nLDL was blocked by an angiotensin II receptor type 1 antagonist. Our data provide evidence for a new mechanism how increased LDL plasma levels might contribute to enhanced endothelin-1 release in patients with hypercholesterolemia.     

Cellular distribution of endothelin-converting enzyme-1 in human tissues.

Endothelin-converting enzyme-1 (ECE-1) is the key enzyme of endothelin biosynthesis, catalyzing the final processing step. As shown by the targeted disruption of the ECE-1 gene, mature endothelins must be produced at specific sites for normal embryonic development. Therefore, it is important to know the exact pattern of ECE-1 gene expression. In this study we investigated the cellular distribution of ECE-1 in a variety of human tissues by in situ hybridization and immunohistochemistry. Widespread expression of the ECE-1 gene was noted, with a similar distribution pattern for mRNA and protein in normal human tissues, suggesting a major biological role for ECE-1. ECE-1 levels were particularly high in the cardiovascular, reproductive, and endocrine systems. There was strong and consistent labeling for ECE-1 in the vascular endothelial cells of all organs examined and in various nonvascular cells, especially some glandular cells. A large amount of ECE-1 protein and mRNA was detected in the Leydig cells of the testis and in the granulosa and theca cells of the ovary. In the adrenal gland, ECE-1 was detected in the cortex and medulla, with the strongest labeling in the zona glomerulosa. Therefore, ECE-1 may be involved in other systems, such as the regulation of hormone secretion, rather than exclusively generating ET-1 from its precursor. These results point out the potential side effects of ECE-1 inhibitors that are currently under development for treatment of cardiovascular diseases. (J Histochem Cytochem 47:447-461, 1999)     

Induction of endothelin-converting enzyme-1 in gastric mucosal injury by idomethacin

Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide produced from a 39-amino acid biologically inactive peptide, big ET-1, by the action of endothelin-converting enzyme-1 (ECE-1). We investigated gastric mucosal expression of ECE-1 during a 16 h course of inflammatory responses associated with gastric mucosal injury caused by indomethacin. The extent of gastric mucosal damage reached a maximum 4 h following the drug, and was accompanied by a 3.9-fold enhancement in the expression of ECE-1 activity and a significant elevation in ET-1 (4.5-fold), TNF-alpha (11.3-fold), and apoptosis (29.9-fold). A 37.2% decrease in the severity of lesion 16 h following the drug was associated with a 44.5% reduction in the mucosal expression of ECE-1 activity and a decline in TNF-alpha (64%), ET-1 (65.2%), and apoptosis (72.3%). The results demonstrate that gastric mucosal injury by indomethacin is associated with up-regulation of ECE-1 expression, which leads to the enhancement of ET-1 production, induction of TNF-alpha, and triggering apoptotic events that disrupt gastric mucosal homeostasis.     

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.     

Distribution of endothelin-converting enzyme-1 and neutral endopeptidase in human endometrium.

The expression of endothelin-1 (ET-1), which has been proposed to have a potential autocrine/paracrine role, varies during the menstrual cycle, and therefore, ET-1 may be involved in the cyclic change of the human endometrium. However, neither the synthesis nor the degradation of ET-1 in the endometrium has been determined in detail. We investigated endothelin-converting enzyme-1 (ECE-1), which converts big-ET-1 to active ET-1, and neutral endopeptidase (NEP), which cleaves and inactivates ET-1 in human endometrium in vivo and in vitro. Western blot analysis demonstrated that the change in the expression of ECE-1 during the menstrual cycle differed from that of NEP in the endometrium. ECE-1 was expressed by endometrial epithelial cells, whereas NEP was predominantly expressed by stromal cells in vivo and in vitro. In conclusion, our results suggest that spacio-temporal expression of two endopeptidases, ECE-1 and NEP, involved in the synthesis and degradation of ET-1, might regulate ET-1 action in human endometrium.     

Synthesis and biological activity of novel potent endothelin-converting enzyme-1 inhibitors

Through directed screening of metalloprotease inhibitors, CGS 30084 (1) has been identified as a potent endothelin-converting enzyme-1 (ECE-1) inhibitor in vitro (IC50 = 77 nM). Herein we report the syntheses and biological activities of analogues derived from this lead, based on modifications of the biphenyl moiety. Compound 10, the thioacetate methyl ester prodrug derivative of compound 6m, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.     

The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg x kg(-1) x day(-1)) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels. L-NAME administration (45 mg x kg(-1) x day(-1)) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.     

Endothelin-converting enzyme-1 expression in acute and chronic liver injury in fibrogenesis

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl₄ administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl₄-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12?h after acute CCl₄-intoxication. Tissue levels of ET-1, ET_A- and ET_B receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl₄-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.     

Synthesis and biological activity of potent heterocyclic thiol-based inhibitors of endothelin-converting enzyme-1

Directed screening of metalloprotease inhibitors identified CGS 30084 (1) as a potent inhibitor of endothelin-converting enzyme-1 (ECE-1) in vitro (IC(50)=77 nM). Herein we report the syntheses and biological activities of analogues containing modified biphenyl moieties, bearing heterocyclic proximal rings. Compound 20, the thioacetate ethyl ester prodrug derivative of compound 19a, was found to be an orally active and potent inhibitor of ECE-1 activity in rats.     

Heterodimerization of endothelin-converting enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.     

Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound zinc-metallopeptidase that is related to neprilysin in amino acid sequence. A major in vivo function of ECE-1 is the proteolytic conversion of big endothelin-1 to endothelin-1, one of the most potent vasconstricting peptides known. Although ECE-1 was once thought to be specific for the processing of endothelin precursors, it is now known that the enzyme hydrolyzes a number of peptide hormones. We have incorporated knowledge gained from recent studies of ECE-1 substrate specificity to aid the design of internally-quenched fluorescent substrates derived from bradykinin. The best of these substrates, (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(2, 4-dinitrophenyl), is hydrolyzed by ECE-1 with a k(cat)/K(m) value of 1.9 x 10(7) M(-1) s(-1), making it the most sensitive substrate yet described for ECE-1. The substrate is suitable for the rapid, continuous assay of the enzyme using a microplate format in a fluorescence plate reader, thereby simplifying both the purification of ECE-1 and the characterization of its inhibitors. It is demonstrated that (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(2, 4-dinitrophenyl) is also a substrate for neprilysin, but is hydrolyzed 10-fold more efficiently by ECE-1, making this substrate selective for ECE-1. Furthermore, this synthetic peptide is a poor substrate for the matrix metalloproteinases.     

Up-regulation of endothelin-converting enzyme-1 in gastric mucosal inflammatory responses to Helicobacter pylori lipopolysaccharide

Endothelin-1 (ET-1) is a vasoactive peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1). We investigated gastric mucosal expression of ECE-1 during a 10-day course of inflammatory responses associated with acute gastritis elicited by Helicobacter pylori lipopolysaccharide. The ECE-1 activity was associated with microsomal fraction and the level of its expression reflected the extent of mucosal inflammatory involvement. The histologic pattern of inflammation reached a maximum on the 4th day following the lipopolysaccharide and was accompanied by a 4.1-fold enhancement in the expression of ECE-1 activity and a significant elevation in ET-1 (3.1-fold), TNF-alpha (8.8-fold), and apoptosis (11.6-fold). A 41.5% decrease in the severity of mucosal inflammation by the 10th day following the lipopolysaccharide was reflected in a 62.3% reduction in the mucosal expression of ECE-1 and a decline in TNF-alpha, ET-1, and apoptosis. Thus, H. pylori infection causes up-regulation of gastric mucosal ECE-1 expression, which leads to the enhancement of ET-1 production, induction of TNF-alpha, and triggering the apoptotic events that exacerbate the inflammatory process.     

Angiotensin-converting enzyme inhibitor therapy prevents upregulation of endothelin-converting enzyme-1 in failing human myocardium

In this study, we investigated the role of the renin-angiotensin system in expression of the endothelin system in atrial myocardium of patients with congestive heart failure. Atrial myocardium of control patients without angiotensin-converting enzyme (ACE) inhibitor therapy and heart failure patients without or with ACE inhibitor therapy undergoing aorto-coronary bypass surgery was studied. Endothelin-converting enzyme-1 (ECE-1) expression and endothelin-1 peptide level was upregulated in myocardium of heart failure patients without ACE inhibition. ACE inhibitor therapy prevented upregulation of ECE-1 and endothelin-1 in failing myocardium. Prepro-endothelin-1 and endothelin receptor A expression were not affected by heart failure. Endothelin receptor B was downregulated in heart failure patients. Our data demonstrate an upregulation of ECE-1 mRNA expression in failing human myocardium. Inhibition of the renin-angiotensin system by ACE inhibitor treatment prevents upregulation of ECE-1, suggesting that angiotensin II regulates ECE-1 expression in vivo.