Purification, structural characterization, and myotropic activity of endothelin from trout, Oncorhynchus mykiss.

Endothelin (ET) from a nontetrapod species has never been characterized, either structurally or biologically. A single molecular form of trout ET with 21-amino-acid residues was isolated in pure form from an extract of the kidney of the steelhead trout, Oncorhynchus mykiss and its primary structure established as Cys-Ser-Cys-Ala-Thr-Phe-Leu-Asp-Lys-Glu10-Cys-Val-Tyr-Phe-Cys-His- L eu-Asp-Ile-Ile20-Trp. This amino acid sequence shows only three substitutions (Ala4-->Ser, Thr5-->Ser, and Phe6-->Trp) compared with human ET-2, demonstrating that the structure of the peptide has been well conserved during evolution and that the pathway of posttranslational processing of preproendothelin in the trout is probably similar to that in mammals. Synthetic trout ET produced concentration-dependent constrictions of isolated rings of vascular tissue from trout efferent branchial artery (EBA; pD2 = 7. 90 +/- 0.06, n = 5), caeliacomesenteric artery (pD2 = 8.03 +/- 0. 04, n = 4), anterior cardinal vein (ACV; pD2 = 8.57 +/- 0.25, n = 4), and rat abdominal aorta (AO; pD2 = 8.86 +/- 0.08, n = 7). Trout and rat vessels were more sensitive to mammalian ET-1 than to trout ET (pD(2) for human ET-1 in: EBA = 9.12 +/- 0.14; ACV = 9.90 +/- 0.15; AO = 8.86 +/- 0.08), but there was no significant difference in the maximum tension produced by either peptide in these vessels.

[Endothelin and arterial hypertension]. / Endotelina a nadcisnienie tetnicze.

Endothelins (ETs) are peptides of 21 amino acids synthesized and released by variety of cells. Endothelin (now this peptide is called endothelin-1 (ET-1)) was isolated and identified in 1988 by Yanagisawa et al. Following studies revealed two other isoforms of endothelin': Endothelin-2 (ET-2) and endothelin-3 (ET-3). All of them bind to two types of receptors (A and B (ET-A r, ET-Br). ET-A r are responsible for concentration mediating. Two subtypes of ET-B r are known. ET-B1 r mediates vasorelaxation; ET-B2 vasoconstriction. ETs (especially ET-1) have variety of biological actions but the most important are vasoconstrictor and mitogenic action. Through these two mechanism ETs may participate in the pathogenesis and/or in the maintenance of hypertension in both experimental animal models and human essential hypertension. The intravenous infusion of synthetic ET induces a long-lasting elevation of blood pressure in experimental animals and in healthy humans. Number of studies have shown enhanced responses to ET in hypertensive subjects but decreased responses have also been reported. Similarly, plasma levels of ET-1 are either normal or elevated in experimental and human essential hypertension. Numerous investigators have suggested an interaction between ET and angiotensin-converting enzyme inhibitors through the renin-angiotensin system or through the accumulation of endogenous bradykinin. Also calcium antagonists of different classes prevent endothelin-induced contractions. Endothelin- converting enzyme inhibitor (phosphoramidon) and ET-A/B r antagonists (bosentan, BQ-123, FR139317) may have potential role as vasodilators in the treatment of hypertension.

Purification of endothelin from a conditioned medium of cardiac fibroblastic cells using beating rate assay of myocytes cultured in a serum-free medium.

A conditioned medium from cardiac fibroblastic cells stimulated the beating of quiescent cardiac myocytes cultured in a serum-free medium. The aim of this study was to isolate and characterize the myocyte beat-stimulating activity of the conditioned medium of fibroblastic cells. Cardiac myocytes and fibroblastic cells were isolated individually from neonatal rats. The fibroblastic cells were grown in a growth medium until they became confluent, then serum-free conditioned medium was obtained from them. For the beating-rate assay, the cardiac myocytes were cultured in a completely serum-free medium. The beat-stimulating factor of myocytes in the conditioned medium was purified by reverse-phase liquid chromatographies and gel filtration, and was characterized by measuring the molecular weight of the activity and a pharmacological antagonistic study. The beat-stimulating activity in the conditioned medium was purified into two active fractions. Both of the activities have a molecular weight of 2.5 kDa, and the activities were abolished similarly by FR139317, an endothelin type-A receptor antagonist. These results indicate that cardiac fibroblastic cells secrete endothelin and that this may contribute in part to the functional abnormalities of the heart in patients with myocardial fibrosis.

Arrhythmogenic action of endothelin peptides in isolated perfused whole hearts from guinea pigs and rats.

The arrhythmogenic actions of endothelin peptides were studied in isolated perfused hearts from guinea pigs and rats. Digoxin-induced ectopic ventricular complexes were partially antagonized by phosphoramidon, an endothelin-converting enzyme inhibitor. On the contrary, these rhythm disturbances were potentiated by big endothelin-1 in isolated perfused whole hearts from guinea pigs. Endothelin-1, when infused through the coronary circulation at a concentration of 10(-10) mol/l, produced an increase in coronary perfusion pressure without altering the heart rate and contractility in the isolated perfused hearts of rats. However, ventricular ectopic complexes occurred when the rise in coronary perfusion pressure reached the peak value. BQ 485, an endothelin-A receptor antagonist, at a concentration of 10(-6) mol/l, completely blocked the vasoconstrictor and arrhythmogenic effects of endothelin-1. In BQ 485-pretreated rat hearts, endothelin-1 produced a fall in coronary perfusion pressure and a slight positive inotropic response which could be blocked by NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor. BQ 485 at the same concentration also caused a significant reduction in the duration but not the onset of ventricular ectopic complexes in the guinea pig isolated perfused heart induced by digoxin. These results were taken as evidence of the arrhythmogenic action of endothelin peptides and their possible participation in the ventricular dysrhythmia induced by digoxin.

Identification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.

Identification and localization of endothelin-1 and its receptors in human fetal jaws.

In the mouse, disruption of the endothelin-1 (ET-1) gene causes severe craniofacial deformities, including mandibular hypoplasia. Since the phenotype of ET-1-deficient mice shows features in common with inherited human mandibulofacial dysostosis, we investigated the presence of ET-1 and its receptors in human fetal craniofacial tissues of 9- to 12-week-old fetuses. We found that ET-1 is immunolocalized in the epithelial cells of the oral cavity. Radioligand binding studies indicate the presence of elevated concentrations of both ETA and ETB receptors in membranes derived from fetal jaws. Using autoradiography, 125I-ET-1 binding sites were shown to be localized within the embryonic mandibular process of the oral cavity, where they were confined to the mesenchymal-derived osteogenic cells. Our data suggest a role for ET-1 in the development of the human mandible.

Processing of proendothelin-1 by human furin convertase.

Endothelin-1 (ET-1) is the most potent vasoactive peptide known to date. The peptide is initially synthesized as an inactive precursor (proET-1) which undergoes proteolysis at specific pairs of basic amino acids to yield bigET-1. Production of ET-1 then proceeds by cleavage of bigET-1 by the endothelin converting enzyme (ECE). Here, we demonstrate that the in vitro cleavage of proET-1 by furin, a mammalian convertase involved in precursor processing, produced bigET-1. Upon further processing, bigET-1 was converted to biologically active ET-1. Furthermore, we demonstrate that the furin inhibitor, decanoyl-Arg-Val-Lys-Arg chloromethylketone, abolished production of ET-1 in endothelial cells.

High yield expression and purification of human endothelin-1.

A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%.

Specific heparin fractions suppress endothelin-1 production in cultured human umbilical vein endothelial cells.

Vascular endothelial cells produce endothelin-1, a peptide with potent vasoconstrictor and mitogenic properties. Heparin suppresses thrombin-stimulated endothelin-1 production in endothelial cells; this is consistent with its reported effect of lowering blood pressure. Since heparin is a heterogeneous mixture of glycosaminoglycans, we examined the effects of different fractions of heparin in suppressing endothelin-1 production in cultured endothelial cells. Heparin fractions differing in size and in antithrombin III affinity were prepared. The results show that the suppressive effect of heparin is independent of these properties of size and antithrombin III affinity. Heparin sulfate suppressed endothelin-1 production to a similar level as heparin. These experiments were conducted in a complete culture medium in the absence of added thrombin. To assess the role of endogenous thrombin in the medium on this process, we tested the effects of hirudin, a specific thrombin inhibitor peptide, on suppression of endothelin-1. Hirudin, like heparin, binds to the anion-binding exosite of thrombin. Hirudin alone, and combined with heparin, suppressed endothelin levels to the same extent as heparin. These experiments demonstrate that the suppressive effect of heparin is the result of its binding to the traces of thrombin in the culture medium, preventing stimulation of endothelin-1 production. This study supports the hypothesis that the functional thrombin receptor may participate in the stimulation of endothelin-1 by thrombin.

Biosynthesis of human endothelins in transformants expressing cDNAs for human prepro-endothelins.

To understand the biosynthesis of the human ET family, three kinds of cloned cDNA for human prepro-endothelin-1 (prepro-ET-1), prepro-ET-2, and prepro-ET-3 were stably expressed in Chinese hamster ovary cells (CHO-K1). Immunoreactive (ir-) ET polypeptides in the culture media of the transformants were purified by reverse-phase high-performance liquid chromatography (HPLC) coupled with sandwich-type enzyme immunoassay (EIA). Amino acid sequencing and FAB mass spectrometry of the purified ir-ET polypeptides revealed the presence of human big ET-1 (1-38), big ET-2 (1-38), and big ET-3 (1-41)NH2 as intermediate forms. These results directly revealed the biosynthetic pathways of three human ETs at the peptide level.

Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli.

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.

Novel bioactive compounds from Actinomycetes: a short review (1988-1992).

Novel secondary metabolites continue to be isolated from Actinomycetes. Their biological activities and chemical structures show a wide range of diversity. This short review provides information on the compounds isolated between 1988 and 1992, and highlights interesting substances discovered during screening.

In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg-->Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.

Localization of endothelin-1-like immunoreactivity in human placenta.

We examined the distribution of endothelin-1-like immunoreactivity in human placenta, using the immunoperoxidase technique. A specific polyclonal antibody to endothelin-1 was raised in rabbits, which recognized endothelin-1 and its precursor molecule, big endothelin. Immunoperoxidase staining revealed that endothelin-1-like immunoreactivity was widely distributed in the placenta. Endothelin-1-like immunoreactivity was present in endothelial cells of capillaries of the microvilli and in small- and medium-sized arteries and veins. The distribution of endothelin-1-like immunoreactivity was similar to the distribution of Factor VIII-related antigen, which stains endothelial cells. The nature of endothelin in the human placenta was further examined with cultured umbilical vein endothelial cells. Endothelial cells released endothelin-like material into the culture medium. The amount of endothelin-like material varied directly with time of incubation and the amount of fetal calf serum in the culture medium. Fractionation of the endothelin-1-like material by reversed-phase high-performance liquid chromatography (HPLC) and quantitation by radioimmunoassay (RIA) revealed that endothelin-like immunoreactivity co-eluted with endothelin-1 but not with big endothelin-1. We conclude that endothelin-1-like immunoreactivity is widely distributed in vascular endothelium of the human placenta. These data are compatible with a role for endothelin as an autocrine or paracrine modulator of vascular tone in the human placenta.