Endothelin converting enzyme of bovine carotid artery smooth muscles

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme in vascular smooth muscle. Like cultured endothelial cells, noncultured vascular smooth muscle homogenate of bovine carotid arteries, converts human big ET- 1 to ET-1 at pH 3.0, pH 5.0 and pH 7.0, and the apparent ratio of these three activities is about 6:5:1, respectively. Peptides generated during incubation of the homogenate and big ET- 1 at the three pHs were identified as ET- 1 by radioimmunoassay and high performance liquid chromatography. The two acid enzymes are in the cytosol (103,000xg sup) and are inhibited by pepstatin A, while the neutral enzyme is sensitive to EDTA or phosphoramidon; 73% of the neutral enzyme activity was membrane-bound and the remainder (27%) cytosolic.     

Phosphoramidon-sensitive endothelin-converting enzyme in the cytosol of cultured bovine endothelial cells

Neutral metalloproteases with endothelin-1 (ET-1) converting activity were detected in membranous and cytosolic fractions of cultured endothelial cells (EC) from bovine carotid artery in a ratio of 5:1, respectively. The cytosolic enzyme specifically and quantitatively converts big ET-1 to ET-1 (Km = 10.7 microM), but does not convert big ET-3. Like the membranous enzyme, the cytosolic enzyme is only active at pH 6.5-7.5, and is competitively inhibited by phosphoramidon (Ki = 0.79 microM). The apparent molecular weight of the cytosolic enzyme is about 540 kD, which is 5-6 times greater than that of the membranous enzyme. These results indicate the presence of two types of phosphoramidon-sensitive neutral ET-converting enzyme in vascular EC.     

Identification and characterization of endothelin converting activity in cultured bovine endothelial cells

Using a specific and sensitive radioimmunoassay (RIA) for the carboxyl terminal tail of endothelin (ET) (His16-Trp21), we have confirmed the presence of the converting activity from synthetic human big ET-1 to ET-1 in the homogenate of cultured bovine aortic endothelial cells. The optimal pHs for the converting activities were found at pH 3.0 and pH 7.0. The activity at pH 3.0 was completely inhibited by pepstatin A, whereas the activity at pH 7.0 was not affected by known various protease inhibitors except EDTA and EGTA. When the products from big ET-1 were analyzed on an ODS and a CN columns, only ET-1 was detected at pH 7.0, but various ET-like immunoreactivities other than ET-1 were detected at pH 3.0. These findings strongly suggest that mature ET-1 is formed from big ET-1 in the endothelial cells by a metal-dependent neutral protease.     

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.     

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.     

Analysis of big endothelin-1 digestion by cathepsin D

Digestion of big endothelin (ET)-1 by cathepsin D, which is the only substantially identified protease showing ET converting enzyme activity, was characterized. Increased doses of cathepsin D showed decrease of immunoreactive (ir-) ET produced from big ET-1. Time course of big ET-1 conversion showed marked increase of ir-ET in a relatively short period, but further incubation resulted in the decrease of ir-ET. Incubation at various pHs with different doses of cathepsin D revealed that low doses of cathepsin D yielded the maximum production of ir-ET at pH 3.5-4.0, but higher doses of cathepsin D showed a bimodal curve of ir-ET production, which may be due to degradation of ir-ET. HPLC analysis revealed that cathepsin D cleaves Asn18-Ile19 bond in addition to Trp21-Val22 bond of big ET-1. These data suggests cathepsin D is not a physiological endothelin converting enzyme.     

N-ethylmaleimide differentiates endothelin converting activity by two types of metalloproteinases derived from vascular endothelial cells

We have recently found that cultured vascular endothelial cells (ECs) contain two types of metalloproteinases which convert big endothelin-1 (big ET-1) to endothelin-1 (ET-1) via a single cleavage between Trp21 and Val22. In the present study, two enzymes were clearly differentiated by using sulfhydryl blocking reagents and anion-exchange HPLC. As reported, the converting activity of the membrane fraction of ECs was specifically inhibited by phosphoramidon. N-ethylmaleimide (NEM) markedly enhanced the apparent converting activity of the membrane fraction. This enhancement was not due to the direct action on the converting enzyme, but rather to inhibition of the degradation of big ET-1 and/or ET-1. In contrast, the converting activity of the cytosolic fraction was abolished by NEM treatment. Effects of phosphoramidon and NEM on converting activities of both fractions were confirmed after anion-exchange HPLC of each fraction, using a COSMOGEL QA column. Our results provide new information on two types of metalloproteinases which convert big ET-1 to ET-1, in vascular ECs.     

Phosphoramidon-sensitive endothelin-converting enzyme in vascular endothelial cells converts big endothelin-1 and big endothelin-3 to their mature form.

Incubation of big endothelin-3 (big ET-3(1-41)) with the membrane fraction obtained from cultured endothelial cells (ECs) resulted in an increase in immunoreactive-ET (IR-ET). This increasing activity was markedly suppressed by phosphoramidon, which is known to inhibit the conversion of big ET-1(1-39) to ET-1(1-21). Reverse-phase HPLC of the incubation mixture of the membrane fraction with big ET-3 revealed one major IR-ET component corresponding to the elution position of synthetic ET-3(1-21). When the cultured ECs were incubated with big ET-3, a conversion to the mature ET-3, as well as an endogenous ET-1 generation, was observed. Both responses were markedly suppressed by phosphoramidon. By the gel filtration of 0.5% CHAPS-solubilized fraction of membrane pellets of ECs, the molecular mass of the proteinase which converts big ET-1 and big ET-3 to their mature form was estimated to be 300-350 kDa. Phosphoramidon almost completely abolished both converting activities of the proteinase. We conclude that the above type of phosphoramidon-sensitive metalloproteinase functions as an ET-converting enzyme to generate the mature form from big ET-1 and big ET-3 in ECs.     

Characterization of phosphoramidon-sensitive metalloproteinases with endothelin-converting enzyme activity in porcine lung membrane

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 μM) 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 α-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.     

Cultured bovine endothelial cells convert big endothelin isopeptides to mature endothelin isopeptides

The incubation of big endothelin-1 (big ET-1), big ET-2 or big ET-3 with cultured bovine endothelial cells (ECs) resulted in their conversions to mature endothelins (ETs). These conversions apparently exhibited Michaelis-Menten kinetics as a function of each big ET isopeptide. The conversions of big ETs were abolished by phosphoramidon. These results indicate that vascular endothelium can convert exogenous big ET-1 to mature ET-1 through a phosphoramidon-sensitive metalloprotease, and that this enzyme has also high affinities for big ET-2 and big ET-3.     

Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.     

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.     

Inhibition of biological actions of big endothelin-1 by phosphoramidon

Endothelin (ET)-1 and big ET-1 both caused contraction of isolated porcine coronary arteries, but the potency of big ET-1 was 1/100-1/200 that of ET-1. These responses were independent of the vascular endothelium. Phosphoramidon blocked the vasoconstriction caused by 30 nM big ET-1, but was ineffective on the action of 0.3 nM ET-1. Also in vivo, phosphoramidon had no effect on the ET-1-induced pressor actions, but blocked the pressor and airway-contractile responses to big ET-1 in rats and/or guinea pigs. Thus, it is likely that the vascular responses to exogenous big ET-1 are at least in part due to its conversion to ET-1 by a phosphoramidon-sensitive ET converting enzyme(s) in the vascular smooth muscle in vitro and in vivo.     

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.     

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.     

Characterization of the contractile and relaxant action of the endothelin-1 precursor, big endothelin-1, in the isolated rat basilar artery

The presence of functional endothelin converting enzyme (ECE) activity in basilar artery ring segments was investigated by measuring the contractile and relaxant effects of big endothelin (ET)-1. Under resting tension conditions cumulative application of big ET1-1 elicited a concentration-related contraction with the concentration-effect curve (CEC) shifted to the right against ET-1 by a factor of 31 and 29 in segments with the endothelium intact or mechanically removed, respectively. Preincubation with the ET(A) receptor antagonist, BQ123, induced an apparently parallel rightwards shift without affecting the maximum contraction. This shift was more pronounced for ET-1 than for big ET-1. With the putative ECE inhibitor phosphoramidon (10(-3) M) in the bath a small rightwards shift of the CEC for big ET-1 was observed in control segments and a more marked one in de-endothelialized segments. In segments precontracted with prostaglandin (PG) F(2alpha) big ET-1 induced a significant although transient relaxation whereas ET-1 did not. However, in the presence of BQ123 both ET-1 and big ET-1 elicited concentration-related relaxation with a significantly higher maximum effect obtained with big ET-1. The potency was 13 fold higher for ET-1, which is markedly less than that found for contraction. The results, therefore, suggest 1) the presence of functional ECE-activity in the rat basilar artery wall, and 2) differences in the functional ECE activity located in the endothelium and media.     

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.     

Do the structures of big ET-1 and big ET-3 adopt a similar overall fold? Consequences for endothelin converting enzyme specificity

Big ET-1 and big ET-3 are precursor peptides which render endothelin-1 (ET-1) and endothelin-3 (ET-3) relatively unreactive and resistant to proteolytic cleavage. Big ET-1 is cleaved in vivo by ECE-1 (endothelin-converting enzyme), and big ET-3 is also cleaved but apparently to a significantly lesser extent by this enzyme. To shed light on the relation between structure and function, circular dichroism (CD) spectroscopy and homology modeling were used to determine whether big ET-1 and big ET-3 adopt similar secondary and tertiary structures. Analyses of the CD spectra and thermal denaturation indicate they have similar secondary structures and thermal stabilities. Superposition of the modeled coordinates of both peptides indicates that they can adopt the same overall fold except in the C-terminal residues, 34-38 in big ET-1 and 34-41 in big ET-3. This region corresponds to an area of complete sequence heterogeneity between the two peptides. A model has been developed which has a loop for residues 27-30 (HVVP in big ET-1), which have previously been demonstrated to be essential for eliciting efficient hydrolysis of the W21-V22 bond in big ET-1 and which have the sequence QTVP in big ET-3. Differences in affinity between big ET-1 and big ET-3 for ECE-1 thus appear to be due solely to sequence variations in the local region of the cleavage site.     

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.     

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.