Osamu Shinmi

Structure-activity relationships of endothelin: importance of the C-terminal moiety

The vasoconstrictor activities of various forms of derivatives of endothelin (ET) were characterized in vitro by measuring the contraction of porcine coronary artery strips. The removal of the C-terminal Trp21 reduced the molar potency of the peptide by nearly 3 orders of magnitude. The removal of amino acid residues from the C-terminus of ET(1-20) further attenuated the activity. Replacement of Trp21 with D-Trp, reduction and carboxamidomethylation of the four Cys residues, or cleavage at Lys9 by lysyl endopeptidase all lowered the potency approximately 200 fold. While both native ET and [D-Trp21]ET induced a very slow and sustained vasoconstriction, the other derivatives of ET listed above showed a much more rapid kinetics of vasoconstriction. These results indicate that the C-terminal Trp of ET is especially important for the potent and extremely long-lasting vasoconstrictor activity characteristic to ET.

Conversion of big endothelin-1 to 21-residue endothelin-1 is essential for expression of full vasoconstrictor activity: Structure-activity relationships of big endothelin-1

Summary The vasoconstrictor activities of porcine big endothelin-1 (big ET-1), a 39-residue intermediate predicted from cDNA sequence analysis, and of its shorter derivative, big ET-1 [1–25], were characterized in vitro by measuring the contraction of porcine coronary artery strips. Synthetic big ET-1 [1–39] and big ET-1 [1–25] induced a slow developing, long-lasting, and strong vasoconstriction as in the case of 21-residue ET-1. However, the contractile molar potencies of big ET-1 [1–39] and big ET-1 [1–25] were approximately 140-and 50-fold lower than that of ET-1, respectively. These results indicate that the conversion of big ET-1 to “mature” ET-1 is essential for the expression of the full vasoconstrictor activity, suggesting the physiological importance of the unusual proteolytic processing catalyzed by the putative “ET converting enzyme.”

Endothelin-3 is a novel neuropeptide: Isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain

The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system.

Presence of endothelin-1 in porcine spinal cord: Isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.

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.

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.

Purification and characterization of putative endothelin converting enzyme in bovine adrenal medulla: Evidence for a cathepsin D-like enzyme

A specific and sensitive assay has been established for measurement of endothelin converting activity in a tissue extract. This assay is based on measuring endothelin-1 generated from big endothelin-1 by endothelin converting enzyme (ECE) with radioimmunoassay using an endothelin C-terminal specific antibody. By using this assay, we purified and characterized ECE in bovine adrenomedullary chromaffin granules ECE was purified over 3,000 times by a combination of DEAE, hydrophobic and gel filtration chromatography. A molecular weight of ECE was estimated to be approximately 30,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ECE had three major components with estimated molecular weights of 45,000, 30,000 and 15,000 like bovine spleen cathepsin D. ECE had a pH optimum at 3.5 and was inhibited by pepstatin. These results strongly suggest that ECE is a cathepsin D-like aspartic protease.

Characterization of endothelin converting enzyme activities in soluble fraction of bovine cultured endothelial cells

Endothelin converting enzyme activities in the soluble fraction of cultured bovine aortic endothelial cells were characterized. The two major endothelin converting enzyme activities were eluted from a hydrophobic chromatography column and the elution profile of the endothelin converting enzyme activities was the same as that of cathepsin D activities. These activities had a same pH optimum at pH 3.5 and were effectively inhibited by pepstatin A. Furthermore, anti-cathepsin D antiserum absorbed these activities as well as cathepsin D activity. Immunoblotting analysis using the antiserum showed the major active fractions have immunostainable components of identical molecular weights with cathepsin D. From these results, we concluded that the major endothelin converting activities in the soluble fraction of endothelial cells are due to cathepsin D. In addition to these cathepsin D activities, a minor endothelin converting enzyme activity with an optimum pH at 3.5 was found, which does not have angiotensin I generating (cathepsin D) activity from renin substrate and needs much higher concentrations of pepstatin A to inhibit the activity than cathepsin D.

Characteristics of binding of endothelin-1 and endothelin-3 to rat hearts. Developmental changes in mechanical responses and receptor subtypes.

Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced positive inotropic effects on electrically stimulated left atria and increased the frequency of spontaneously beating right atria of adult rats. The potency of the inotropic effect of ET-1 was greater than that of ET-3, but the potencies of the chronotropic effects of ET-1 and ET-3 were not significantly different. In the neonatal atria, ET-1 and ET-3 also induced positive inotropic and chronotropic responses. ET-1 and ET-3 showed weak or no cardiotonic effects on the adult ventricles, whereas they caused marked positive inotropy in the neonatal ventricles. The characteristics of binding sites for ET-1 and ET-3 were very similar between the atria and the ventricles of the rat neonate. Saturation and competition binding experiments have shown that neonatal cardiac membranes from both atria and ventricle have two distinct binding sites for endothelin, that is, a low-affinity and a high-affinity site. ET-1 was found to bind to the low-affinity sites with a significantly lower Kd than ET-3, whereas the estimated Kd values for ET-1 and ET-3 at the high affinity sites were similar. In contrast, the binding sites in adult atria were different from those of the ventricles: only a single binding site for both ET-1 and ET-3 was detected. Adult atrial membranes, on the other hand, had two distinct binding sites similar to those of neonatal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.