Phosphoramidon, a metalloproteinase inhibitor, suppresses the secretion of endothelin-1 from cultured endothelial cells by inhibiting a big endothelin-1 converting enzyme

Time-dependent secretion of immunoreactive-endothelin (IR-ET) from cultured porcine aortic endothelial cells was markedly suppressed by phosphoramidon is due to proteinase inhibitor. Analysis of the culture supernatant with or without phosphoramidon by reverse-phase high performance liquid chromatography confirmed that the suppression of IR-ET secretion by phosphoramidon is due to a decreae in secretion of endothelin-1-like materials. The secretion of the C-terminal fragment (CTF, 22-39)-like materials of big ET-1 was also decreased by phosphoramidon, whereas there was an increased secretion of big ET-1-like materials. These data strongly suggest that phosphoramidon suppresses the secretion of ET-1 from cultured endothelial cells by inhibiting the conversion of big ET-1 to ET-1. It is most likely that phosphoramidon-sensitive metalloproteinase is responsible for the processing of big ET-1 in vascular endothelial cells.     

Phosphoramidon inhibits the generation of endothelin-1 from exogenously applied big endothelin-1 in cultured vascular endothelial cells and smooth muscle cells

When cultured porcine aortic endothelial cells (ECs) were incubated with porcine big endothelin-1 (bit ET-1(1-39)), there was a time-dependent increase in immunoreactive (IR)-ET in the culture supernatant, in addition to an endogenous IR-ET release fron the cells. Reverse-phase HPLC of the culture supernatant revealed one major IR-ET component corresponding to the elution position of synthetic ET-1, thereby indicating that the additional increase in IR-ET was due to the conversion of big ET-1 to mature ET-1(1-21). Phosphoramidon, a metalloproteinase inhibitor, strongly suppressed this increase in IR-ET as well as the endogenous IR-ET release. Cultured vascular smooth muscle cells (VSMCs) also released IR-ET. The apparent conversion of exogenously applied big ET-1 to ET-1 and its inhibition by phosphoramidon were observed using cultured VSMCs, although the enzyme inhibitor did not influence the basal secretion of IR-ET from VSMCs. These results suggest that both cultured ECs and VSMCs can generate ET-1 from exogenously applied big ET-1 via action of the same type of phosphoramidon-sensitive metalloproteinase, which is also involved in the endogenous ET-1 generation in 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.     

Conversion of big endothelin-1 to endothelin-1 by two-types of metalloproteinases of cultured porcine vascular smooth muscle cells

Incubation of big endothelin-1 (big ET-1, 1-39) with the membrane fraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon, a metalloproteinase inhibitor. When the incubation was performed in the presence of N-ethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentation was abolished by phosphoramidon. The pH profile for IR-ET generation in the presence of NEM was apparently distinct from that observed in the absence of NEM. Reverse-phase HPLC of the incubation mixture with or without NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (1-21). When the cultured VSMCs were incubated with big ET-1, a conversion to the mature ET-1 was observed. This ET-1 generation from exogenously applied big ET-1 was markedly inhibited by the addition of phosphoramidon, although the inhibitor did not influence the basal secretion of ET-1-like materials. These results suggest the presence of two types of metalloproteinases, which can generate ET-1, in VSMCs. The possibility that ET-1 functions in an autocrine manner to control the cardiovascular system warrants further attention.     

Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells

Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.     

Phosphoramidon inhibits the intracellular conversion of big endothelin-1 to endothelin-1 in cultured endothelial cells

Effects of various protease inhibitors on the conversion of big endothelin (ET)-1 to ET-1 in cultured endothelial cells were analyzed. A metal protease inhibitor, phosphoramidon, decreases the amount of ET-1 and increase that of big ET-1 released. This effect is dose-dependent and not nonspecific. When the contents of ET-1 and big ET-1 in the cells after culturing in the medium with or without phosphoramidon were measured, the ratio of ET-1: big ET-1 in the cells was 3.3 : 1 and phosphoramidon inverted the ratio in the cells to 1 : 3.5. These data strongly suggest that a phosphoramidon-sensitive protease converts big ET-1 to mature ET-1 intracellularly.     

Analysis of endothelin related peptides in culture supernatant of porcine aortic endothelial cells: evidence for biosynthetic pathway of endothelin-1

We investigated the molecular forms of endothelin (ET) related peptides in culture supernatant of porcine aortic endothelial cells by high performance liquid chromatography coupled with radioimmunoassays for ET related peptides. We isolated and sequenced a C-terminal peptide (big ET-1(22-39] of big ET-1(1-39) and its N-terminal truncated form (big ET-1(23-39] in addition to ET-1(1-21) and its oxidized form, [Met7 (0)]ET-1(1-21). The total contents of the two C-terminal peptides of big ET-1(1-39) are approximately equal to those of ET-1(1-21) and its oxidized form on a molar basis in the culture supernatant. Furthermore, we isolated big ET-1(1-39) although its content is approximately 2% of that of ET-1(1-21). These results strongly suggest that ET-1(1-21) and big ET-1(22-39) are generated from big ET-1(1-39) by specific processing between Trp21-Val22.     

Characterization of endothelin secretion by vascular endothelial cells

The characterization of mechanisms that regulate ET-LP secretion from bovine adrenal cortical capillary endothelial cells (ACE) in culture was performed by developing radioimmunoassays that distinguish between ET1-21 (AbET1-21) and ET1-39 (AbET1-39). The conditioned media (DMEM) content of ET-like immunoreactivity (ET1-21LI) increased from 50 to 350 pg/ml over a 24 h period. Addition of 10% calf serum or 0.1% BSA enhanced ET1-21LI release 2-3 fold. Authenticity of ET1-21LI was examined using reversed phase liquid chromatography. All ET1-21LI co-eluted with authentic ET-1. Examination of ET1-39IR by liquid chromatography revealed two peaks of immunoreactivity, one co-eluting with authentic ET22-39 and a later running peak co-eluting with authentic ET1-39. Neither ET1-21LI nor ET1-39LI was detected in the extracts of sonicated ACE cells. Treatment of cells with various forms of TGF beta significantly augmented ET1-21LI release. These data suggest that ACE secretion of ET-LP in vitro spontaneously and can be enhanced by TGFss. Since neither ET1-21 LI nor ET1-39 LI was detectable detectable in ACE cells it is unlikely that ET-LP are stored prior to their secretion.     

Endothelin-1 is expressed and released by a human endothelial hybrid cell line (EA.hy 926).

A permanent vascular endothelial cell line, EA.hy 926, was shown to express endothelin-1 (ET-1) mRNA and to secrete big ET-1 and ET-1 into culture medium. The concentration of both big ET-1 and ET-1 was significantly increased in EA.hy 926 culture medium by phosphoramidon, a metalloproteinase inhibitor, suggesting that phosphoramidon sensitive protease(s) may be responsible for the degradation of ET-1 and big ET-1. EA.hy 926 cells responded to various regulators of ET-1 similarly as primary human vascular endothelial cells. The production of ET-1 was increased by thrombin and decreased by vasodilators such as atrial natriuretic peptide, brain natriuretic peptide and nitroprusside, and by 8-bromo cyclic GMP and papaverine. This continuous human endothelial hybrid cell line could facilitate studies of regulation of ET-1 production in human endothelial cells, which in primary cultures have limited replication potential.     

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.     

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.     

Conversion of big endothelin-1 by membrane-bound metalloendopeptidase in cultured bovine endothelial cells

We propose a candidate for the "putative" endothelin (ET) converting enzyme in the cultured endothelial cells (ECs) of bovine carotid artery. The enzyme is membrane-bound, soluble in 0.5% Triton X-100, and capable of converting human big ET-1 to ET-1 by a specific cleavage between Trp21 and Val22. The conversion reached 90% after a 5-hr incubation in the presence of DFP, PCMS and pepstatin A, but it was inhibited by EDTA, omicron-phenanthroline or phosphoramidon. The enzyme is very sensitive to pH, and active only between pH 6.6 and pH 7.6. Conversion of big ET-3 by this enzyme was only 1/9 that of big ET-1. From these results, ET-1 converting enzyme in the bovine EC is most likely to be a membrane-bound, neutral metalloendopeptidase, which is much less susceptible to big ET-3.     

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-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.     

Secretory response of endothelin-1 in cultured human glomerular microvascular endothelial cells to shear stress

The shear-induced secretory response of endothelin-1 (ET-1) by human microvascular endothelial cells was studied using paired human glomerular microvascular endothelial cell (HGMEC) cultured monolayers exposed to steady-state laminar shear stress for up to 10 hours. The first cell monolayer was subjected to a shear stress of 0.65 N m-2 and the second, 1.3 N m-2. ET-1 secretion was determined by radioimmunoassay. Over 10 hours of shear, the total cumulative secretion of ET-1 was 237.4 pg/cm2 for the monolayer exposed to 1.3 N m-2 and 143.6 pg/cm2 for the monolayer exposed to 0.65 N m-2. The average ET-1 secretion rate was 20.90 +/- 2.15 and 12.45 +/- 1.05 pg/cm2.h at 0.65 N m-2 and 1.3 N m-2, respectively. The results showed that ET-1 secretion varied with the time of shear in a nonlinear fashion. Although the level of shear stress affected the absolute value of ET-1 cumulative secretion and secretion rate, the major secretion period for both monolayers occurred between 2.0 and 8.0 hours, with the peak secretion rate occurring at approximately 5 hours. Thus, the response of cultured human microvascular endothelial cells to shear stress differed from that of large vessel endothelial cell cultures in terms of ET-1 secretion. In addition to the level of shear stress, the time of shear was also an important determinant of ET-1 secretion. Consequently, the heterogeneity of vascular endothelial cells and the time of shear should both be considered in future research on the secretion of vascular endothelial cell cultures.     

Cardiotrophin-1 stimulates endothelin-1 via gp130 in vascular endothelial cells

Endothelin-1 (ET-1) is a vasoconstricting and mitogenic peptide released from vascular endothelial cells under normal and pathophysiological conditions, and synthesis and secretion of ET-1 are stimulated by cytokines. Cardiotrophin-1 (CT-1) is a new member of the interleukin-6-type cytokines that induce biological actions through the glycoprotein (gp) 130. The present study was designed to determine the presence of CT-1 and the gp130 cytokine system in vascular endothelial cells and to investigate whether CT-1 stimulates synthesis and secretion of ET-1 in the vascular endothelial cells. We first sought to determine gene expression and immunoreactivity of CT-1, gp130 and ET-1 in cultured canine aortic endothelial cells (CAECs) using Northern blot analysis and immunocytochemistry, which revealed the presence of CT-1 and gp130 together with ET-1 in CAECs. CT-1 increased ET-1 gene expression in CAECs, and stimulated ET-1 secretion from CAECs in a dose-dependent manner. Furthermore, inhibition of gp130 by monoclonal antibody attenuated ET-1 secretion from CAECs, suggesting that actions of CT-1 on the secretion of ET-1 are mediated through gp130 receptor system. The present study, therefore, reports the presence of CT-1 and gp130 in vascular endothelial cells and mechanisms of secretion of ET-1 related to this cytokine system.     

Coronary microvascular endothelial cells cosecrete angiotensin II and endothelin-1 via a regulated pathway

Although endothelial cells produce angiotensin II (ANG II) and endothelin-1 (ET-1), it is not clear whether a single cell produces both peptides, with cosecretion in response to stimulation, or whether different subpopulations of endothelial cells secrete one or the other peptide, with secretion in response to different stimuli. Exposure of cultured coronary microvascular endothelial cells to cycloheximide for 60 min had no effect on ANG II or ET-1 secretion. This result suggested the existence of a preformed intracellular pool of ANG II and ET-1, which is a precondition for regulated secretion. Exposure of endothelial cells to isoproterenol, high extracellular potassium, or cadmium, all of which stimulate peptide secretion via different signaling pathways, significantly (P > 0.001) increased the secretion of both ANG II and ET-1 in a cell size-dependent manner. Sodium nitroprusside and S-nitroso-N-acetyl penicillamine significantly (P > 0.001) decreased ANG II and ET-1 secretion, whereas N(omega)-nitro-L-arginine-methyl ester enhanced it. The similar regulation of ANG II and ET-1 secretion and the presence of both peptides around individual endothelial cells indicate that the autocrine/paracrine regulation of cardiovascular function by endothelial cells is accomplished via cosecretion of ANG II and ET-1.     

Nucleotide sequence of endothelin-1 cDNA from rabbit endothelial cells.

A cDNA encoding rabbit endothelin-1 (ET-1) was isolated by plaque hybridization from a rabbit inferior vena caval endothelial cell lambda gt11 cDNA library using human ET-1 cDNA as the hybridization probe. DNA sequence analysis indicates that mature 21 amino acid rabbit ET-1 is derived from a 202 amino acid precursor, via a 39 amino acid intermediate 'big' ET-1.