endothelin-1 and phosphoramidon

Topics: Acute Kidney Injury; Animals; Biomarkers; Contrast Media; Endothelin Receptor Antagonists; Endothelin-1; Endotoxemia; Erythropoietin; Fibrosis; Glycopeptides; Heart Failure; Humans; Hypotension; Kidney Failure, Chronic; Peptides, Cyclic; Peritoneal Dialysis; Peritoneum; Prognosis; Recombinant Proteins; Renal Dialysis

The endothelins are a family of three structurally related peptides. Endothelin-1 (ET-1) is formed from the big endothelin by the action of the endothelin converting enzyme. It acts on two types of receptor, ETA and ETB. ET-1 is a powerful vasoconstrictor but also has a number of other effects: positive inotropism and stimulation of cell growth, for example. Endothelin is found in the general circulation but its role is mainly local in maintaining vascular tone. The endothelin system is activated in cardiac failure and increased concentrations of plasma endothelin increased, ET-1 converting enzyme and increased density of endothelin receptors are observed. The action of the endothelin system and its relationships with other neuro-hormonal systems activated in cardiac failure are not fully understood but research is under way which should clarify these mechanisms in the next few years. In view of the properties of endothelin, inhibition of its action might be particularly useful in patients with cardiac failure. Its action can be blocked either by preventing its synthesis by inhibiting the endothelin converting enzyme or by blocking the endothelin receptor. Endothelin receptor blockade is associated with beneficial haemodynamic changes, an action on ventricular remodelling and possibly an improved prognosis. Many substances, either selective for ETA receptors or mixed ETA and ETB receptor blockers, are under development. The benefits of these products will require confirmation by large scale clinical trials.

Topics: Cardiac Output, Low; Chronic Disease; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glycopeptides; Humans; Metalloendopeptidases

A critical processing step in endothelin biosynthesis is the conversion of the intermediate "big endothelin" to its biologically active product catalysed by endothelin converting enzyme (ECE). In this commentary we discuss critically the cellular location, structure, and activity of the isoforms of ECE. The current evidence supporting a metallopeptidase ECE as the physiological regulator of endothelin production is described. Its sensitivity to inhibition by the fungal metabolite phosphoramidon and subsequent cloning of the enzyme indicate it to be a type II integral membrane protein homologous with neural endopeptidase-24.11 (E-24.11), the major neuropeptide-degrading ectoenzyme in brain and other tissues. Unlike E-24.11, however, ECE exists as a disulphide-linked dimer of subunit M(r) 120-130 kDa and is not inhibited by other E-24.11 inhibitors such as thiorphan. Alternative splicing produces two forms of ECE with distinct N-terminal tails. These isoforms of ECE-1 show similar specificity converting big endothelin-1 (ET-1) to ET-1 but big ET-2 and big ET-3 are converted much less efficiently. This suggests that additional forms of ECE remain to be isolated. Immunocytochemical studies indicate a predominant cell-surface location for ECE-1, like E-24.11. This is consistent with the conversion of exogenous big ET-1 when administered in vivo and the inhibition of this event by phosphoramidon. However, mature ET-1 can be detected in intracellular vesicles in endothelial cells, suggesting that some processing occurs in the constitutive secretory pathway. This may be mediated by ECE-2, a recently cloned member of the E-24.11/ECE family which has an acidic pH optimum. Selective inhibitors of ECE may have therapeutic applications in cardiovascular and renal medicine.

Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Cloning, Molecular; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Humans; Metalloendopeptidases; Molecular Sequence Data; Neprilysin; Protease Inhibitors; Protein Precursors; Substrate Specificity

In the present study, we hypothesized that endotoxemia produces metalloendopeptidase (MEPD)-dependent generation of endothelin-1 (ET-1) and alters NOS expression correlating with p38-mitogen-activated protein kinase (MAPK) phosphorylation in thoracic aorta. Male Sprague-Dawley rats (350-400 g) were subjected to two groups randomly; sham-treated (N = 10) and lipopolysaccharide (LPS)-treated (N = 10) (E. coli LPS 2 mg/kg bolus + 2 mg/kg infusion for 30 min). The animals in each group were further subdivided into vehicle and MEPD inhibitor phosphoramidon (1 mg/kg bolus, PHOS)-treated groups. LPS produces a significant decrease in mean arterial pressure (MAP) at 2 h post endotoxemia that was blocked by PHOS. PHOS attenuated LPS-induced increase in tumor necrosis factor-alpha (TNF-alpha) concentration at 2- and 24 h post-LPS administration. LPS significantly elevated plasma concentrations of ET-1 at 2- and 24 h post endotoxemia. An upregulated preproET-1 expression following both LPS and MEPD inhibition was observed in thoracic aorta at 2 h post treatment. PHOS effectively blocked conversion of preproET-1 to ET-1 in thoracic aorta locally at 24 h post treatment in endotoxic rats. PHOS inhibited LPS-induced upregulation of inducible NOS (iNOS), downregulation of endothelial NOS (eNOS) and elevation of NO byproducts (NOx) in thoracic aorta. PHOS also blocked LPS-induced upregulated p38-MAPK phosphorylation in thoracic aorta at 24 h post endotoxemia. The data revealed that LPS induces MEPD-sensitive inflammatory response syndrome (SIRS) at 2- and 24 h post endotoxemia. We concluded that inhibition of MEPD not only decreases the levels of ET-1 but also simultaneously downregulates protein expression of iNOS and phosphorylated p38-MAPK while increasing eNOS in thoracic aorta during SIRS in endotoxemia. We suggest that MEPD-dependent ET-1 and NO mechanisms may be involved in endotoxemia-induced altered p38-MAPK phosphorylation.

Topics: Animals; Aorta, Thoracic; Blood Pressure; Cytokines; Endothelin-1; Endotoxemia; Glycopeptides; Immunoblotting; Lipopolysaccharides; Male; Metalloendopeptidases; Models, Biological; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Rats, Sprague-Dawley; Time Factors; Up-Regulation

It was previously found that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs (1-31). In the present study, human plasma concentrations of ET-1 (1-31) and ET-1 were examined and the effect of synthetic ET-1 (1-31) on the proliferation of cultured human mesangial cells (HMCs) was investigated. The proliferative effect of ET-1 (1-31) was evaluated from the [3H]-thymidine uptake. The activity of extracellular signal-regulated kinase (ERK) and DNA binding activity of activator protein-1 were determined by using an in-gel kinase assay and gel mobility shift assay, respectively. Immunoreactive ET-1 (1-31) was detectable in plasma, but the level was slightly lower than that of ET-1. ET-1 (1-31) increased [3H]-thymidine incorporation in HMCs to a degree similar to that induced by ET-1. ET-1 (1-31) also activated ERK1/2. Inhibition of protein kinase C and ERK kinase caused a reduction of ET-1 (1-31)-induced ERK1/2 activation. The ERK1/2 activation was followed by an increase in transcription factor activator protein-1 DNA binding activity. These findings suggest that ET-1 (1-31) is a bioactive peptide in humans and ET-1 (1-31) itself stimulates HMC proliferation.

Topics: Adult; Cell Division; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Drug Interactions; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Enzyme Activation; Enzyme Inhibitors; Female; Glomerular Mesangium; Glycopeptides; Humans; Male; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinases; Peptide Fragments; Protein Kinase C; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Transcription Factor AP-1

Endothelin-1 generated by the vascular endothelium contributes to basal vascular tone and blood pressure in healthy humans. Plasma concentrations of endothelin-1, which are elevated in chronic renal failure (CRF), may contribute to increased vascular tone.. We investigated the contribution of endogenous and exogenous endothelin-1 to the maintenance of vascular tone in patients with CRF (creatinine > or = 200 mumol/liter) and in age- and sex-matched healthy subjects. In a series of experiments, we measured forearm vascular responses to intra-arterial norepinephrine (30 to 240 pmol/min), endothelin-1 (5 pmol/min), the selective endothelin A (ETA) receptor antagonist BQ-123 (3 mg/hr), the mixed endothelin-converting enzyme and neutral endopeptidase inhibitor phosphoramidon (30 nmol/min), and the selective neutral endopeptidase inhibitor thiorphan (30 nmol/min).. The maximum reduction in forearm blood flow (FBF) to norepinephrine in CRF (33 +/- 7%) was similar to that in controls (43 +/- 7%, P = 0.53). Endothelin-1 also produced a similar reduction in FBF in CRF (35 +/- 6%) and controls (36 +/- 5%, P = 0.81). BQ-123 increased FBF in CRF (11 +/- 4%) but significantly less than in controls (44 +/- 10%, P = 0.02). Phosphoramidon increased FBF in CRF (68 +/- 20%), again significantly less than in controls (181 +/- 41%, P = 0.001). Thiorphan reduced FBF similarly in CRF (22 +/- 6%) and controls (14 +/- 6%, P = 0.39). Responses to phosphoramidon were substantially greater than to BQ-123.. These studies show that endogenous generation of endothelin-1 contributes to the maintenance of resting vascular tone in patients with CRF, as well as in healthy subjects. Although the contribution of endogenous endothelin-1 to resting vascular tone appears to be reduced in CRF, ETA receptor antagonism, and particularly endothelin-converting enzyme inhibition, should be explored as means by which to reduce vascular tone and blood pressure in patients with CRF.

Topics: Adult; Endothelin Receptor Antagonists; Endothelin-1; Female; Forearm; Glycopeptides; Humans; Injections, Intra-Arterial; Kidney Failure, Chronic; Male; Middle Aged; Norepinephrine; Peptides, Cyclic; Protease Inhibitors; Receptor, Endothelin A; Regional Blood Flow; Thiorphan; Vasomotor System

The importance of endothelin-1 in chronic heart failure (CHF) is unclear. We therefore investigated the effects of endothelin-converting enzyme (ECE) inhibition and endothelin ETA receptor blockade in CHF patients treated with ACE inhibitors. We also compared the function of ETA and ETB receptors in healthy subjects and patients with CHF.. Locally active doses of study drugs were infused into the nondominant brachial artery while forearm blood flow (FBF was measured by venous occlusion plethysmography. In CHF patients (n = 10), phosphoramidon (a combined ECE and neutral endopeptidase inhibitor) and BQ-123 (an ETA receptor antagonist) increased FBF by 52 +/- 10% (P = .0006) and 31 +/- 6% (P = .002), respectively, and thiorphan (a selective neutral endopeptidase inhibitor) reduced FBF by 15 +/- 5% (P = .0007). Forearm vasoconstriction to endothelin-1 (an ETA and ETB receptor agonist) was significantly blunted in CHF patients compared with control subjects (both n = 10; CHF versus control subjects, P < .001), whereas vasoconstriction to sarafotoxin S6c (an ETB receptor agonist) was significantly enhanced in CHF patients compared with control subjects (both n = 10; CHF versus control subjects. P < .05).. ECE inhibitors and ETA receptor antagonists may be useful as vasodilator agents in CHF patients already receiving treatment with an ACE inhibitor. Both ETA and ETB receptors can mediate agonist-induced vasoconstriction in healthy subjects and patients with CHF, but further studies are required to clarify the contribution of each receptor subtype in mediating the effects of endogenous endothelin-1.

Topics: Aged; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Aspartic Acid Endopeptidases; Brachial Artery; Chronic Disease; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Female; Glycopeptides; Heart Failure; Humans; Male; Metalloendopeptidases; Middle Aged; Neprilysin; Peptides, Cyclic; Protease Inhibitors; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Regional Blood Flow; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Viper Venoms

Endothelin-1 is a potent endothelium-derived vasoconstrictor and pressor peptide with uniquely sustained activity. We have examined the contribution of endogenously-generated endothelin-1 to the maintenance of basal vascular tone in healthy subjects. In these studies, on separate occasions, a combined inhibitor of endothelin converting enzyme (ECE) and neutral endopeptidase (NEP), phosphoramidon, a selective inhibitor of NEP, thiorphan, and a selective ETA receptor antagonist, BQ-123, were given via the brachial artery. Big endothelin-1, the precursor to endothelin-1, caused a slow onset dose-dependent forearm vasoconstriction, the magnitude of which was consistent with about 10% conversion to mature endothelin-1 in the forearm. Vasoconstriction to big endothelin-1 was abolished by co-infusion of phosphoramidon, whereas vasoconstriction to endothelin-1 was unaffected. Phosphoramidon caused progressive vasodilatation when infused alone, with blood flow increasing by 37% at 90 min (P = 0.02), whereas thiorphan caused vasoconstriction, consistent with NEP inhibition exerting its major effect on degradation of constrictor peptides, such as angiotensin and endothelin-1. Vasoconstriction to endothelin-1 was completely abolished by coinfusion of BQ-123, and BQ-123 alone produced progressive forearm vasodilatation, with blood flow increasing by 64% after 60 min (P = 0.001). These results demonstrate that endogenous production of endothelin-1 acts to sustain vascular tone in humans and indicate that ECE inhibitors and endothelium receptor antagonists may have therapeutic potential as vasodilators.

Topics: Adult; Blood Circulation; Endothelin-1; Endothelins; Glycopeptides; Humans; Male; Muscle Tonus; Peptides, Cyclic; Protease Inhibitors; Protein Precursors; Thiorphan; Vascular Resistance; Vasoconstriction; Vasodilation; Vasomotor System

Endothelin-1 is an endothelium-derived vasoconstrictor peptide, possibly involved in the pathophysiology of cardiovascular disease. We examined the contribution of endogenously generated endothelin-1 to maintenance of peripheral vascular tone in healthy subjects by local intraarterial administration of an inhibitor of endothelin converting enzyme, phosphoramidon, and of a selective endothelin receptor A antagonist, BQ-123. Brachial artery infusion of local doses of proendothelin-1, the precursor to endothelin-1, caused a slow-onset dose-dependent forearm vasoconstriction which was abolished by co-infusion of phosphoramidon. Phosphoramidon did not affect responses to endothelin-1. Phosphoramidon caused slow-onset vasodilatation when infused alone, with blood flow increasing by 37% at 90 min (p = 0.03). Vasoconstriction to endothelin-1 was abolished by co-infusion of BQ-123 (p = 0.006), with forearm blood flow tending to increase. Infusion of BQ-123 alone caused progressive vasodilatation, with blood flow increasing by 64% after 60 min (p = 0.007). These results show that endogenous production of endothelin-1 contributes to the maintenance of vascular tone. Endothelin converting enzyme inhibitors and receptor antagonists may have therapeutic potential as vasodilators.

Topics: Adult; Anti-Bacterial Agents; Blood Pressure; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Forearm; Glycopeptides; Heart Rate; Humans; Infusions, Intra-Arterial; Male; Neprilysin; Peptides, Cyclic; Protein Precursors; Regional Blood Flow; Vasoconstriction; Vasodilation

Atherosclerosis is the primary cause of CAD and cerebrovascular disease. Endothelin (ET)-1 is a vasoconstrictive peptide implicated in Atherosclerosis pathology. Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin. Reported inhibitors of ECE-1 and their IC(50) values were retrieved from literature and their structures were docked with the parent protein using the Molegro virtual docker. The obtained MolDock scores of each of the compounds are hereby reported and are subject to graphical analysis in conjunction with their respective IC(50) values to characterize potent inhibitors. A search was then run in the ZINC database for compounds with similar properties. Potent inhibitors with higher Dock scores and better Ranking were isolated and are reported.

Topics: Aspartic Acid Endopeptidases; Databases, Protein; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Humans; Hydrogen Bonding; Inhibitory Concentration 50; Metalloendopeptidases; Molecular Dynamics Simulation; Protein Binding; Structure-Activity Relationship

This study investigated endothelin-1 (ET-1) production induced by vascular endothelial growth factor (VEGF) in two different vascular wall cell types.. We analyzed the effect of endothelin-converting enzyme-1 (ECE-1) inhibitor and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) on VEGF-induced ET-1 expression using real-time polymerase chain reaction and enzyme-linked immunosorbent assay in human umbilical vein endothelial cells and aortic smooth muscle cells.. In human umbilical vein endothelial cells, both phosphoramidon, an inhibitor of ECE-1, and TIMP-2 decreased VEGF-induced ET-1 production. In aortic smooth muscle cells, TIMP-2 suppressed VEGF-induced ET-1 production, but phosphoramidon did not influence ET-1 concentration in culture.. VEGF-induced ET-1 production may be MMP-2- or ECE-1-dependant in endothelial cells; however, in smooth muscle cells, ET-1 expression appears to be induced by MMP-2 only.

Topics: Aorta; Aspartic Acid Endopeptidases; Endothelial Cells; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Glycopeptides; Humans; Metalloendopeptidases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Tissue Inhibitor of Metalloproteinase-2; Umbilical Veins; Vascular Endothelial Growth Factor A

Using perfused hearts from streptozotocin-induced long-term diabetic rats, we studied the coronary vasoconstrictor effect of the endothelin-1 (ET-1) precursor big ET-1 and also whether this response was modulated by N(epsilon)-(carboxymethyl)lysine (CML; a representative advanced glycation end product that is implicated in the pathogenesis of diabetic vasculopathy). The big ET-1-induced vasoconstriction (a) developed more rapidly (i.e., was greater in the first 30 min) in the diabetic group than in the age-matched controls, and (b) in each group was largely suppressed by phosphoramidon [nonselective endothelin-converting enzyme (ECE)/neutral endopeptidase (NEP) inhibitor] or CGS35066 (selective ECE inhibitor), but not by thiorphan (selective NEP inhibitor). The ET-1 release occurring after treatment with big ET-1, which was greater in diabetic coronary arteries than in the controls, was reduced by CGS35066. The dose-response curve for ET-1 was shifted to the left in the diabetics, so that at some lower doses of ET-1 the vasoconstriction was greater than in the controls. CML enhanced big ET-1- or ET-1-induced vasoconstriction in the controls, but not in the diabetics. Finally, the plasma level of CML was higher in diabetic than in control rats. These findings suggest (a) that the increased responsiveness to big ET-1 shown by diabetic coronary arteries may be attributable both to a more rapid conversion of big ET-1 to ET-1 (by ECE), allowing it to exert its contractile activity, and to an increased vascular sensitivity to ET-1, and (b) that CML may be at least partly responsible for the diabetes-associated enhancement of big ET-1-mediated coronary vasoconstriction.

Topics: Animals; Aspartic Acid Endopeptidases; Benzofurans; Blood Glucose; Body Weight; Coronary Vessels; Diabetes Mellitus, Experimental; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Heart; Lipids; Lysine; Male; Metalloendopeptidases; Myocardium; Oligopeptides; Organ Size; Organophosphonates; Peptides, Cyclic; Perfusion; Piperidines; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Thiorphan; Vasoconstriction

Big endothelin-1 (ET-1) circulates in plasma but does not bind to ET receptors until converted to ET-1 by smooth muscle converting enzymes. We hypothesized that tissue-specific conversion of [(18)F]-big ET-1 to [(18)F]-ET-1 could be imaged dynamically in vivo within target organs as binding to ET receptors.. [(18)F]-big ET-1 conversion imaged in vivo following infusion into rats using positron emission tomography (PET).. [(18)F]-big ET-1 was rapidly cleared from the circulation (t(1/2)= 2.9 +/- 0.1 min). Whole body microPET images showed highest uptake of radioactivity in three major organs. In lungs and liver, time activity curves peaked within 2.5 min, then plateaued reaching equilibrium after 10 min, with no further decrease after 120 min. Phosphoramidon did not alter half life of [(18)F]-big ET-1 but uptake was reduced in lung (42%) and liver (45%) after 120 min, consistent with inhibition of enzyme conversion and reduction of ET-1 receptor binding. The ET(A) antagonist, FR139317 did not alter half-life of [(18)F]-big ET-1 (t(1/2)= 2.5 min) but radioactivity was reduced in all tissues except for kidney consistent with reduction in binding to ET(A) receptors. In kidney, however, the peak in radioactivity was higher but time to maximum accumulation was slower ( approximately 30 min), which was increased by phosphoramidon, reflecting renal excretion with low conversion and binding to ET receptors.. A major site for conversion was within the vasculature of the lung and liver, whereas uptake in kidney was more complex, reflecting excretion of [(18)F]-big ET-1 without conversion to ET-1.

Topics: Animals; Aspartic Acid Endopeptidases; Autoradiography; Azepines; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Activation; Fluorine Radioisotopes; Glycopeptides; Half-Life; Indoles; Infusions, Intravenous; Kidney; Lung; Male; Metalloendopeptidases; Molecular Imaging; Molecular Probe Techniques; Positron-Emission Tomography; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Tissue Distribution; Whole Body Imaging

Circulating levels of endothelin (ET)-1 and endogenous ET(A)-mediated constriction are increased in human aging. The mechanisms responsible are not known.. Investigate the storage, release, and activity of ET-1 system in arteries from young and aged Fischer-344 rats.. After NO synthase inhibition (L-NAME), thrombin contracted aged arteries, which was inhibited by endothelial denudation, ET(A) receptor antagonism (BQ123), and ECE inhibition (phosphoramidon, SM19712) or by inhibiting exocytosis (TAT-NSF, N-ethylmaleimide-sensitive factor inhibitor). Thrombin did not cause endothelium-dependent contraction of young arteries. In aged but not young arteries, thrombin rapidly increased ET-1 release, which was abolished by endothelium denudation or TAT-NSF. L-NAME did not affect ET-1 release. ET-1 immunofluorescent staining was punctate and distinct from von Willebrand factor (VWF). VWF and ET-1 immunofluorescent intensity was similar in young and aged quiescent arteries. Thrombin rapidly increased ET-1 staining and decreased VWF staining in aged but had no effect in young aortas. After L-NAME, thrombin decreased VWF staining in young aortas. NO donor DEA-NONOate (1 to 100 nmol/L) reversed thrombin-induced exocytosis in young (VWF) but not aged L-NAME-treated aortas (VWF, ET-1). Expression of preproET-1 mRNA and ECE-1 mRNA were increased in aged compared to young endothelium. BigET-1 levels and contraction to exogenous BigET-1 (but not ET-1) were also increased in aged compared to young arteries.. The stimulated exocytotic release of ET-1 is dramatically increased in aged endothelium. This reflects increased reactivity of exocytosis, increased expression and storage of ET-1 precursor peptides, and increased expression of ECE-1. Altered endothelial exocytosis of ET-1 and other mediators may contribute to cardiovascular pathology in aging.

Topics: Age Factors; Aging; Animals; Aorta, Thoracic; Aspartic Acid Endopeptidases; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Exocytosis; Fluorescent Antibody Technique; Glycopeptides; Hydrazines; In Vitro Techniques; Mesenteric Arteries; Metalloendopeptidases; N-Ethylmaleimide-Sensitive Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Peptides, Cyclic; Rats; Rats, Inbred F344; Receptor, Endothelin A; RNA, Messenger; Sulfonamides; Sulfonylurea Compounds; Thrombin; Up-Regulation; Vasoconstriction; von Willebrand Factor

Activation of the endothelin (ET) system has been implicated in the pathogenesis of retinal ischemic disease. Although ET-1, the predominant endogenous isoform of ET, has been shown to cause constriction of retinal vessels, the expression and functional significance of its synthesis and the involved specific ET receptors in retinal arterioles remain unknown. The authors examined the roles of ET(A) and ET(B) receptors and of endothelin-converting enzyme (ECE)-1 in ET-1-induced vasomotor responses of single retinal arterioles.. To exclude systemic confounding effects, porcine retinal arterioles were isolated for vasoreactivity and molecular studies.. Isolated and pressurized retinal arterioles developed basal tone and constricted in a manner dependent on concentration to ET-1. ET-1 precursor big ET-1 elicited time-dependent vasoconstriction over 20 minutes, which was blocked by the ECE-1 inhibitor phosphoramidon. ET(A) receptor antagonist BQ123 inhibited most (approximately 90%) of vasoconstrictions to ET-1 and big ET-1. ET(B) receptor agonist sarafotoxin also elicited concentration-dependent constriction of retinal arterioles but with significantly less potency than ET-1. ET(B) receptor antagonist BQ788 abolished vasoconstriction to sarafotoxin but only slightly reduced responses to ET-1 and big ET-1. Protein and mRNA expressions of ET(A), ET(B), and ECE-1 were detected in retinal arterioles. Immunohistochemistry revealed ET(A) and ET(B) receptors predominantly in smooth muscle and ECE-1 predominantly in endothelium and smooth muscle.. ET-1 elicits constriction of retinal arterioles predominantly through the activation of smooth muscle ET(A) receptors. Endogenous production of ET-1 from vascular ECE-1 is sufficient to evoke ET(A) receptor-dependent constriction in retinal arterioles.

Topics: Animals; Arterioles; Aspartic Acid Endopeptidases; Blotting, Western; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Female; Fluorescent Antibody Technique, Indirect; Glycopeptides; Male; Metalloendopeptidases; Muscle, Smooth, Vascular; Peptides, Cyclic; Protease Inhibitors; Receptor, Endothelin A; Receptor, Endothelin B; Retinal Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine; Vasoconstriction; Vasomotor System; Viper Venoms

Endothelin-1 and norepinephrine are involved in myocardial ischemia/reperfusion injury. The aim of this study was to investigate the role of endogenously generated endothelin-1 in ischemia/reperfusion-induced norepinephrine overflow and cardiac dysfunction using a nonselective prototype of endothelin-converting enzyme (ECE) inhibitor, phosphoramidon, and a selective ECE inhibitor, SM-19712 (4-chloro-N-[[(4-cyano-3-methyl-1-phenyl-1H-pyrazol-5-yl)amino]carbonyl]benzenesulfonamide, monosodium salt). According to the Langendorff technique, isolated Sprague-Dawley rat hearts were subjected to 40-min global ischemia followed by 30-min reperfusion. Phosphoramidon and SM-19712 were perfused 30 min before ischemia and during reperfusion. Endothelin-1 level in left ventricle was increased by ischemia/reperfusion. This increase in left ventricular endothelin-1 level was suppressed by treatment with SM-19712. SM-19712 significantly improved ischemia/reperfusion-induced cardiac dysfunction such as decreased left ventricular developed pressure and dP/dt(max) and increased left ventricular end diastolic pressure. In addition, this agent suppressed excessive norepinephrine overflow in the coronary effluent from the post-ischemic heart. In contrast, treatment with phosphoramidon further enhanced left ventricular endothelin-1 level and norepinephrine overflow, and significantly worsened cardiac dysfunction after ischemia/reperfusion. These responses such as exaggerated norepinephrine overflow and the cardiac dysfunction observed after ischemia/reperfusion were markedly suppressed in the presence of a selective endothelin ET(A) receptor antagonist, ABT-627 [2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid]. These findings indicate that cardiac endothelin-1 production is enhanced by ischemia/reperfusion, and this endogenously increased endothelin-1 is involved in post-ischemic norepinephrine overflow and cardiac dysfunction via the activation of endothelin ET(A) receptors.

Topics: Animals; Aspartic Acid Endopeptidases; Atrasentan; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Male; Metalloendopeptidases; Myocardial Ischemia; Myocardial Reperfusion Injury; Norepinephrine; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Sulfonamides; Sulfonylurea Compounds

We previously found that endothelin-1(1-31) (ET-1(1-31)) exhibited a pro-arrhythmogenic effect in isolated rat hearts. In this study, we further investigated the effects of ET-1(1-31) on a cell viability and observed [Ca(2+)](i) in cultured cardiomyocytes. Cultured neonatal rat cardiomyocytes were treated with 0.1, 1, and 10 nM ET-1(1-31) for 24h in the presence or absence of ET(A) receptor antagonist (BQ(123)) or phosphoramidon, a NEP/ECE inhibitor. Cell injury was evaluated by supernatant lactate dehydrogenase (LDH) assay, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. Cell viability was assessed by MTT assay. [Ca(2+)](i) was measured with Fluo-3/AM under a laser confocal microscope. 1) ET-1(1-31) dose-dependently increased LDH release and decreased cell viability. 2) LDH and MDA levels were significantly elevated and SOD activity decreased after administration of 1 nM ET-1(1-31) for 24h, and these changes were markedly attenuated by 1 uM BQ(123). 3) Exposure to 10 nM ET 1(1-31) caused a continuous increase in [Ca(2+)](i) to cultured beating cardiomyocytes and termination of [Ca(2+)](i) transient within 6 min, and this change was reversed by 1 uM BQ(123) and attenuated by 0.5 mM phosphoramidon. These results suggest that ET-1(1-31) could cause cell injury, and that the effect of ET-1(1-31) on [Ca(2+)](i) transients is mainly mediated by ET(A) receptor and partially attributed to the conversion of ET-1(1-31) to ET-1(1-21).

Topics: Animals; Animals, Newborn; Aspartic Acid Endopeptidases; Calcium Signaling; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; L-Lactate Dehydrogenase; Malondialdehyde; Metalloendopeptidases; Microscopy, Confocal; Myocytes, Cardiac; Peptide Fragments; Peptides, Cyclic; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Superoxide Dismutase; Time Factors

Phosphoramidon blocks the formation of endothelin-1 (ET-1), a proinflammatory mediator implicated in the pathogenesis of a variety of lung diseases. To determine whether phosphoramidon can ameliorate pulmonary inflammation, our laboratory undertook a series of experiments involving treatment of hamsters with either intraperitoneal (i.p.) or aerosolized phosphoramidon prior to induction of acute lung injury by intratracheal administration of lipopolysaccharide (LPS). The results indicate that phosphoramidon significantly reduces LPS-induced pulmonary inflammation as measured by lung histology, neutrophil content of bronchoalveolar lavage (BAL) fluid, percent tumor necrosis factor receptor 1 (TNFR1)-labeled BAL macrophages, and alveolar septal cell apoptosis. In additional experiments, i.p. administration of a novel endothelin A receptor anatgonist (HJP272) similarly decreased BAL neutrophils, whereas i.p. administration of either ET-1, or its precursor peptide, "big" ET-1, had the opposite effect. These findings support further evaluation of phosphoramidon and other ET-1 suppressors as potential treatments for human inflammatory lung disease.

Topics: Animals; Apoptosis; Cricetinae; Endothelin A Receptor Antagonists; Endothelin-1; Glycopeptides; Lipopolysaccharides; Lung; Mesocricetus; Receptors, Tumor Necrosis Factor, Type I; Respiratory Distress Syndrome

The purpose of the present work was to investigate whether conversion of exogenous applied big-endothelin-1 (Big-ET-1) as well as the basal release and mRNA levels of endothelin-1 (ET-1) is altered by ethanol consumption in the rat carotid. The measurement of the contraction induced by Big-ET-1 served as an indicative of functional endothelin (ET)-converting enzyme (ECE) activity. Cumulative application of exogenous Big-ET-1 elicited a concentration-related contraction with the concentration-response curve shifted to the right when compared to ET-1. In endothelium-intact rings, phosphoramidon (1 mmol/l), a nonselective ECE/neutral endopeptidase (NEP) inhibitor, produced a rightward displacement of the concentration-response curves and reduced the maximal contractile response to Big-ET-1. However, in endothelium-denuded rings phosphoramidon reduced the maximum contraction for Big-ET-1 but did not alter the potency when compared to the curves obtained in the absence of the inhibitor. Ethanol consumption for 2, 6, or 10 weeks reduced the contractile effect elicited by Big-ET-1 in carotid rings with intact endothelium when compared to control or isocaloric rings. However, no differences on Big-ET-1-induced contraction were observed after endothelial denudation. On the other hand, ethanol consumption increased ET-1-induced contraction. Finally, chronic ethanol consumption did not alter either the mRNA levels for pre-pro-ET-1 nor the basal release of ET-1. The present findings show that chronic ethanol consumption does not alter the mRNA levels for ET-1 or its basal release in the rat carotid. Moreover, ethanol intake reduces the contraction induced by exogenously applied Big-ET-1 in carotid rings with intact endothelium, a fact that might be the result of a reduced conversion of this peptide by ECE on its mature active peptide ET-1.

Topics: Alcohol Drinking; Animals; Aspartic Acid Endopeptidases; Carotid Arteries; Central Nervous System Depressants; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Ethanol; Glycopeptides; Male; Metalloendopeptidases; Protease Inhibitors; Rats; Rats, Wistar; RNA, Messenger; Time Factors; Vasoconstriction

Endothelin (ET)-1(1-21) is known to play an important role in the pathogenesis of acute ischemic arrhythmia. In the present study, we attempted to determine whether administration of ET-1(1-31) would result in arrhythmia in perfused isolated rat hearts. Forty-eight Sprague-Dawley rats weighing approximately 250-350 g were randomized into 6 groups. Heart was isolated and perfused in a Langendorff mode. The effects of ET-1(1-31) on arrhythmia, heart rate, coronary flow, and heart function were analyzed. Perfusion with 1 nM ET-1(1-31) resulted in frequent ventricular ectopic beats (VEBs) and ventricular tachycardia (VT). Overall VEB was 128.0 (approximately 66.0-1015.0), and the arrhythmia score (AS) was 2.18 +/- 0.87; both were significantly higher than those of the control group (P < 0.01). Pretreatment with perfusion of 10 nM of the ETA-receptor antagonist BQ(123) markedly attenuated the occurrence of VEB and VT induced by ET-1(1-31). AS in 10 nM BQ123 group was significantly lower than that in 1 nM ET-1(1-31) group (P < 0.01). The arrhythmia induced by 1 nM ET-1(1-31) was partially but significantly reduced by phosphoramidon (1 microM), a neutral endopeptidase/ET-converting enzyme inhibitor. ET-1(1-31) per se caused arrhythmia in perfused isolated rat hearts. This arrhythmogenic action is in part mediated by ET(A) receptor and may be attributed mainly to the conversion of ET-1(1-31) to ET-1(1-21.).

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Endothelin-1; Glycopeptides; In Vitro Techniques; Male; Peptides, Cyclic; Perfusion; Protease Inhibitors; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Endothelin

Synthetic big endothelin-1 (ET-1), a 39-residue precursor of ET-1, has been reported to elicit potent contractile action on helical strip specimens obtained from the porcine coronary artery, but its molar potency was found to be 140-fold lower than that of ET-1 [Saito, Y., Nakao, K., Mukoyama, M., Imura, H., 1990. Increased plasma endothelin level in patients with essential hypertension. N. Engl. J. Med. 322, 205]. It has been hypothesized that the increased rate of production and/or release of ET-1 from the vascular endothelium may contribute to the pathogenesis of hypertension. However, the effects of big ET-1 in comparison with ET-1 on the macrocirculation and microcirculation of the rat mesentery have not been well documented. Thus, our main purpose for this study was to examine the effects of both big ET-1 and ET-1 to clarify the role of phosphoramidon in inhibiting the conversion of big ET-1 to ET-1, by investigating the systemic blood pressure, microvascular blood flow velocity, and diameters of arterioles and venules of the rat mesentery. For this purpose, two groups of experiments were performed. In these experiments, the mesentery was arranged for in situ intravital microscopic observation under transillumination. In the first group of experiments, intravenous cumulative injections of big ET-1 or ET-1 were infused through a catheter inserted into the right jugular vein. Infusion of big ET-1 (1-8 nmol/kg) elicited a long-lasting significant pressor effect. Infusion of big ET-1 (1-2 nmol/kg) elicited a significant dose-dependent increase in the microvascular blood flow velocity both in arterioles (20-30 microm) and venules (30-40 microm). Microvascular diameters exhibited a slight but significant vasodilator effect. However, the infusion of big ET-1 (4-8 nmol/kg) elicited a dose-dependent significant decrease in the blood flow velocities, and diameters returned to control measurements. The administration of ET-1 (0.25-2 nmol/kg) induced a dose-dependent significant decrease in the blood flow velocity of arterioles and venules, and their diameters exhibited a vasoconstrictive effect more prominent in arterioles than in venules. In the second group of experiments, cumulative injections of phosphoramidon (30 mg/kg/10 min) were administered 10 min prior to the infusion of big ET-1. Phosphoramidon significantly suppressed the long-lasting significant pressor effect and significantly inhibited the dose-dependent increase and dose-dependent decrease in t

Topics: Animals; Arterioles; Blood Flow Velocity; Blood Pressure; Dose-Response Relationship, Drug; Endothelin-1; Glycopeptides; Infusions, Intravenous; Protease Inhibitors; Rats; Splanchnic Circulation; Venules

The precursor of endothelin-1, big endothelin-1, can be hydrolyzed by chymase to generate endothelin-1 (1-31) in vitro. In the present study, we explored the processes involved in the production of endothelin-1 (1-31) as well as its pharmacodynamic characteristics in the rabbit in vivo. Endothelin-1 (1-31) (1 nmol/kg, injected into the left cardiac ventricle) induced a monophasic increase of mean arterial blood pressure similarly to big endothelin-1 (1-38), whereas endothelin-1 induces a biphasic response. Phosphoramidon, a dual neutral endopeptidase and endothelin-converting enzyme inhibitor, blocked both pressor responses to endothelin-1 (1-31) and big endothelin-1 but not those afforded by endothelin-1. Thiorphan, a neutral endopeptidase inhibitor, markedly inhibited the response to endothelin-1 (1-31) but only weakly reduced that of big endothelin-1. In contrast, CGS 35066, an endothelin-converting enzyme inhibitor, was significantly more efficient against the pressor response to big endothelin-1 than to endothelin-1 (1-31). Furthermore, injection of big endothelin-1 concomitantly with phosphoramidon induced an increase in endothelin-1 (1-31) plasma levels. Finally, intracardiac-administered endothelin-1 (1-31) induced an increase of endothelin-1 plasma levels, which are markedly reduced by phosphoramidon and thiorphan but not by CGS 35066. Our results thus demonstrate that endothelin-1 (1-31) is an alternate intermediate in the production of endothelin-1 after big endothelin-1 administration in the rabbit in vivo.

Topics: Animals; Aspartic Acid Endopeptidases; Benzofurans; Blood Pressure; Endothelin-1; Endothelin-Converting Enzymes; Female; Glycopeptides; Heart Ventricles; Injections; Male; Metalloendopeptidases; Neprilysin; Organophosphonates; Peptide Fragments; Protease Inhibitors; Rabbits; Receptors, Endothelin; Thiorphan

The aim of this study was to determine the physiologic role for endogenous endothelin in the regulation of vascular tone during normal pregnancy and preeclampsia. The vascular sensitivity to endothelin-1 during pregnancy was studied also.. Forearm blood flow was measured by venous occlusion plethysmography during intra-arterial infusion of phosphoramidon, an endothelin-converting enzyme inhibitor, for 60 minutes, which was followed by co-infusion with endothelin-1 for 30 minutes. Three groups were studied: healthy nonpregnant women, normal pregnant women, and women with preeclampsia.. There was a significant increase in forearm blood flow in the nonpregnant group after phosphoramidon infusion alone (73%+/-37%; P<.05). Phosphoramidon did not change forearm blood flow in pregnant subjects. Co-infusion with endothelin-1 significantly decreased forearm blood flow in both the nonpregnant and normal pregnant women (53%+/-7% and 40%+/-11%, respectively; P<.01). No response to endothelin-1 was found among women with preeclampsia.. The vascular sensitivity to endothelin-1 is not altered during normal pregnancy in contrast to preeclamptic pregnancy, where no effect of endothelin-1 was seen. Reduced endothelin dependence during pregnancy might be one mechanism behind the fall in peripheral vascular resistance.

Topics: Adult; Aspartic Acid Endopeptidases; Case-Control Studies; Endothelin-1; Endothelin-Converting Enzymes; Female; Forearm; Glycopeptides; Humans; Metalloendopeptidases; Pre-Eclampsia; Pregnancy; Protease Inhibitors; Regional Blood Flow; Vasomotor System

Leptin, a multifunctional hormone that regulates food intake and energy expenditure, has emerged recently as an important modulator of gastric mucosal responses to Helicobacter pylori infection. We applied the animal model of H. pylori LPS-induced gastritis to investigate the role of endothelin-1 (ET-1) in the mucosal leptin production. We show that the histologic pattern of inflammation reached a maximum on the fourth day following the LPS and was reflected in a marked increase in the mucosal level of ET-1 and leptin. Therapeutic administration of phosphoramidon, an inhibitor of ECE-1 activity, led to a 61.2% decline in the mucosal ET-1 level and a 64.1% reduction in leptin, while the severity of mucosal inflammatory involvement increased by 28.6%. A drop in the level of leptin and the increase in severity of the inflammatory involvement elicited by the LPS was also attained in the presence of ET(A) receptor antagonist BQ610, but not the ET(B) receptor antagonist BQ788. Moreover, administration of ERK inhibitor, PD98059, in the presence of ET(B) receptor antagonist, but not the ET(A) receptor antagonist, caused reduction in the mucosal leptin level. Our findings are the first to implicate ET-1 as a key factor in up-regulation of gastric mucosal leptin-associated H. pylori infection. We also show that the effect of ET-1 on leptin production is a consequence of ET(A) receptor activation.

Topics: Animals; Aspartic Acid Endopeptidases; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gastric Mucosa; Gastritis; Glycopeptides; Helicobacter pylori; Leptin; Lipopolysaccharides; Metalloendopeptidases; Oligopeptides; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Up-Regulation

Endothelin-1 1-31 (ET-1 1-31), a novel member of the endothelin family comprising 31 amino acids and derived from the selective hydrolysis of big ET-1 by chymase, directly activates endothelin receptors or converts to ET-1 1-21 by ET converting enzyme (ECE). The cardiovascular effects of central ET-1 1-31 are not identified. The present study was designed to investigate the cardiovascular actions of ET-1 1-31 within the rostral ventrolateral medulla (RVLM) in anesthetized rats. Bilateral injection of ET-1 1-31 (0.5, 1, and 2 pmol for each side) into the rostral ventrolateral medulla produced an initial pressor and/or a long-lasting hypotensive action but did not affect HR. Unilateral microinjection of 2 and 4 pmol of ET-1 1-31 into the rostral ventrolateral medulla only produced a significant (P < 0.05) transient increase in blood pressure by an average of 13 and 12 mm Hg, respectively, whereas unilateral microinjection of 8 pmol of ET-1 1-31 produced a sustained fall in blood pressure (from 92 +/- 6 to 69 +/- 8 mm Hg, P < 0.05). The transient pressor effect of unilaterally injecting ET-1 1-31 (4 pmol) into the rostral ventrolateral medulla was completely abolished by pretreatment with either ETA receptor antagonist BQ123 (83 +/- 2 versus 84 +/- 5 mm Hg, P > 0.05) or ET converting enzyme inhibitor phosphoramidon (99 +/- 5 versus 99 +/- 7 mm Hg, P > 0.05) but not ETB receptor antagonist IRL1038 (89 +/- 6 versus 96 +/- 7 mm Hg, P < 0.05). In addition, prior injection of phosphoramidon also completely abolished the long-lasting hypotension of intra-RVLM ET-1 1-31 (8 pmol) but did not modify the depressor action of intra-RVLM ET-1 1-21 (from 100 +/- 6 to 76 +/- 8 mm Hg, P < 0.05). In conclusion, the current results suggest that the cardiovascular effects of intra-RVLM ET-1 1-31 might be the result of conversion of ET-1 1-31 to ET-1 1-21 through activation of ETA receptors.

Topics: Animals; Blood Pressure; Endothelin A Receptor Antagonists; Endothelin-1; Glycopeptides; Heart Rate; Male; Medulla Oblongata; Peptide Fragments; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A

Leptin, a multifunctional hormone that regulates food intake and energy expenditure, has emerged recently as an important modulator of inflammatory cascades associated with wound healing. In this study, we applied the animal model of buccal mucosal ulcer to investigate the role of endothelin-1 (ET-1) and leptin in soft oral tissue repair. Using groups of rats with experimentally induced buccal mucosal ulcers we show that ulcer onset was characterized by a marked increase in the mucosal level of ET-1 and leptin. However, while the ET-1 level gradually declined with healing, the mucosal level of leptin increased reaching maximum expression on the 4th day of healing. Therapeutic administration of phosphoramidon, an inhibitor of ECE-1 activity, not only led to a 53.2% drop in the ET-1, but also produced a dose-dependent reduction (up to 50.9%) in the mucosal level of leptin and up to 42.3% decline in the rate of ulcer healing. A marked drop (54.2%) in the mucosal level of leptin and the reduction (46.8%) in the rate of ulcer healing was also attained in the presence of ETA receptor antagonist BQ610 administration, but not the ETB receptor antagonist BQ788. Moreover, administration of ERK inhibitor, PD98059 in the presence of ETB receptor antagonist, but not the ETA receptor antagonist, caused the reduction the mucosal leptin level as well as a decline in the rate of ulcer healing. Our findings are the first to implicate the requirement for both ET-1 and leptin in orderly progression of the events of soft oral tissue repair. We also show that ET-1 is a key factor in up-regulation of leptin production associated with oral mucosal ulcer healing , and that the effect of ET-1 on leptin production is a consequence of ETA receptor activation and subsequent signaling through MAPK/ERK.

Topics: Acetic Acid; Animals; Aspartic Acid Endopeptidases; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Flavonoids; Glycopeptides; Leptin; Metalloendopeptidases; Mitogen-Activated Protein Kinase Kinases; Mouth Mucosa; Oligopeptides; Oral Ulcer; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Time Factors; Up-Regulation; Wound Healing

The effect of hypoxia (24 h) on TNF-alpha-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-alpha- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-alpha (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-alpha-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-alpha- and ET-1-mediated hONA proliferation. PD142893 (1 microM), an ET(A/B) receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-alpha treatment. Also, U0126 (10 microM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-alpha and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-alpha and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.

Topics: Aged; Aged, 80 and over; Aspartic Acid Endopeptidases; Astrocytes; Butadienes; Cell Division; Cell Hypoxia; Cell Line, Tumor; Cells, Cultured; Cycloheximide; Dactinomycin; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Humans; Metalloendopeptidases; Mitogen-Activated Protein Kinases; Nitriles; Oligopeptides; Optic Disk; Protease Inhibitors; Tumor Necrosis Factor-alpha

1. The ductus venosus is actively regulated in the fetus, but questions remain on the presence of a functional sphincter at its inlet. Using fetal sheep (0.6-0.7 gestation onwards), we have examined the morphology of the vessel and have also determined whether endothelin-1 (ET-1) qualifies as a natural constrictor being modulated by prostaglandins (PGs). 2. Masson's staining and alpha-actin immunohistochemistry showed a muscular, sphincter-like formation at the ductus inlet and a muscle layer within the wall of the vessel proper. This muscle cell component increased with age. 3. ET-1 contracted dose-dependently isolated sphincter and extrasphincter preparations of the ductus from term fetus. This ET-1 effect also occurred in the premature, but its threshold was higher. 4. BQ123 (1 microm) caused a rightward shift in the ET-1 dose-response curve, while indomethacin at a threshold concentration (28 nm) tended to have an opposite effect. 5. Big ET-1 also contracted the ductus sphincter but differed from ET-1 for its lesser potency and inhibition by phosphoramidon (50 microm). 6. The ductus sphincter (term and preterm) and extrasphincter (term) released 6-keto-PGF(1alpha) (hence PGI(2)) and, to a lesser degree, PGE(2) at rest and their release increased dose-dependently upon ET-1 treatment. Both basal and stimulated release was curtailed by endothelium removal. 7. BQ123 and phosphoramidon reduced slightly the contraction of ductus sphincter to indomethacin (2.8 microm). 8. We conclude that the ductus contains a contractile mechanism in the sphincter and extrasphincter regions. ET-1 lends itself to a role in the generation of contractile tone and its action may be modulated by prostaglandins.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Canada; Dinoprostone; Dose-Response Relationship, Drug; Drug Synergism; Endothelin-1; Female; Fetus; Gestational Age; Glycopeptides; Indomethacin; Muscle, Smooth, Vascular; Peptides, Cyclic; Pregnancy; Prostaglandins; Sheep; Thromboxane A2; Umbilical Veins; Vasoconstriction; Vena Cava, Inferior

Human chymase produces not only angiotensin II but also endothelin(ET)-1(1-31). We previously reported that ET-1(1-31) had several biological activities in vascular smooth muscle cells. In this study, we investigated the vasoconstrictor effect of ET-1(1-31) on the renal resistance vessels using in vitro microperfused rabbit afferent and efferent arterioles. ET-1(1-31) decreased the lumen diameter of the afferent and efferent arterioles dose-dependently. ET-1(1-31)-induced afferent arteriolar vasoconstriction was not affected by phosphoramidon, an ET converting enzyme inhibitor. ET-1(1-31)-induced renal arteriolar vasoconstriction was inhibited by BQ123, an ETA receptor inhibitor, but not by BQ788, an ETB receptor inhibitor. These results suggest that ET-1(1-31)-induced renal arteriolar vasoconstriction may be mediated by ETA-like receptors.

Topics: Animals; Arterioles; Aspartic Acid Endopeptidases; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Humans; Kidney; Male; Metalloendopeptidases; Muscle, Smooth, Vascular; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Perfusion; Piperidines; Rabbits; Receptor, Endothelin A; Receptors, Endothelin; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents

Endothelin-1-(1-31) is a new bioactive 31-amino-acid-length peptide generated from big endothelin-1 by chymase or other chymotrypsin-type proteases with various pathophysiologic functions. In this study, we have detected the specific and monophasic binding of [125I]endothelin-1-(1-31) in porcine lung membranes. Competition studies of [125I]endothelin-1-(1-31) binding by unlabeled endothelin-1-(1-31), endothelin-1, endothelin-3, and antagonists and agonists of endothelin ET(A) and ET(B) receptors suggest that the binding protein is an endothelin ET(B) or ET(B)-like receptor rather than an endothelin ET(A) receptor in porcine lungs. Kinetic studies showed that the affinity of endothelin-1-(1-31) to its receptor was approximately one order of magnitude lower than that of endothelin-1, and that the specific binding of endothelin-1-(1-31) was about 19% of endothelin-1 binding. The binding of [125I]endothelin-1-(1-31) was extremely slow, slower even than that of endothelin-1, and nearly irreversible. This unique quasi-irreversibility may explain the slow-onset and long-lasting biologic effects of this peptide in vivo.

Topics: Animals; Binding, Competitive; Cell Membrane; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-3; Enzyme Inhibitors; Glycopeptides; Humans; Iodine Radioisotopes; Lung; Metalloendopeptidases; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Radioligand Assay; Receptor, Endothelin B; Receptors, Endothelin; Swine

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of protein kinase C. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Chelerythrine, a protein kinase C inhibitor, relaxed the spasm. However, relaxations to chelerythrine were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that subarachnoid hemorrhage induced vasospasm does not involve functional upregulation of protein kinase C.

Topics: Alkaloids; Animals; Basilar Artery; Benzophenanthridines; Endothelin-1; Enzyme Inhibitors; Glycopeptides; Male; Phenanthridines; Protease Inhibitors; Protein Kinase C; Rabbits; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction; Vasospasm, Intracranial

We tested the hypothesis that metalloendopeptidase inhibition using phosphoramidon during induction of endotoxemia 24 h later would down-regulate the protein expression of myocardial inducible nitric oxide synthase (iNOS) and phosphorylation of p38-mitogen-activated protein kinase (p38-MAPK). Male Sprague-Dawley rats (350-400 g) were randomly divided into sham-treated and LPS-treated groups (Escherichia. coli lipopolysaccharide [LPS] 2 mg/kg bolus + 2 mg/kg infusion for 30 min). The animals in each group were further subdivided into vehicle- and phosphoramidon (1 mg/kg bolus)-treated subgroups. Blood and heart samples were collected at 2- and 24-h postendotoxemia/phosphoramidon treatment. LPS at 2 h after its administration produced a significant decrease in mean arterial pressure that was blocked by phosphoramidon treatment. LPS at 2 and 24 h produced a significant elevation in the concentration of left ventricular endothelin-1 (ET-1) both in heart and plasma as compared with control group. This LPS-induced left ventricular ET-1 elevation at 24 h was significantly reduced by phosphoramidon. No significant alterations were observed in the myocardial protein expression of preproET-1, iNOS, and eNOS at 2 h post LPS. In 24-h post treatment groups phosphoramidon upregulated the expression of myocardial preproET-1 protein both in control and endotoxemic rat groups. Also, LPS-induced upregulated protein expression of myocardial-inducible nitric oxide synthase and increased levels of nitric oxide byproducts at 24 h were blocked by phosphoramidon. Phosphoramidon inhibited LPS-induced down-regulated expression of myocardial endothelial nitric oxide synthase and upregulated p38-MAPK phosphorylation. These results indicated that inhibition of metalloendopeptidase during induction of endotoxemia could regulate the phosphorylation of myocardial p38-MAPK and iNOS protein expression at 24-h post endotoxemia. We concluded that inhibition of metalloendopeptidases during early endotoxemia not only decreased the biosynthesis of ET-1 in heart locally but also simultaneously down-regulated myocardial protein expression of iNOS and p38-MAPK phosphorylation in the later stage of endotoxemia.

Topics: Animals; Blood Pressure; Endothelin-1; Endotoxemia; Glycopeptides; Heart; Lipopolysaccharides; Male; Metalloendopeptidases; Mitogen-Activated Protein Kinases; Models, Biological; Myocardium; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protease Inhibitors; Rats; Rats, Sprague-Dawley

This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II.. Isolated cat papillary muscles were used for force, pH(i), [Na(+)](i) and [Ca(2+)](i) measurements and isolated cat myocytes for patch-clamp experiments.. In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23+/-2% (n=4, P<0.05), [Na(+)](i) by 2.2+/-0.2 mmol/l (n=4, P<0.05), and peak (but not diastolic) Ca(2+) from 0.674+/-0.11 to 0.768+/-0.13 micromol/l (n=4, P<0.05), without affecting pH(i). Force and [Na(+)](i) increase were abolished by inhibition of the Na(+)/H(+) exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na(+)](i) increase was abolished by inhibition of reverse Na(+)/Ca(2+) exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21+/-2%, n=4, P<0.05) and in [Na(+)](i) (2.4+/-0.4 mmol/l, n=4, P<0.05) that were also suppressed by TAK044 and HOE642 were induced by exogenous 5.0 nmol/l endothelin-1. KB-R7943 reverted the endothelin-1 effect on force but not on [Na(+)](i). In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (E(NCX)) to a more negative value (DeltaE(NCX): -10+/-3 and -17+/-5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642.. Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na(+)/H(+) exchanger, increases [Na(+)](i) and changes E(NCX), promoting the influx of Ca(2+) that leads to a positive inotropic effect (PIE).

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Cats; Electrophysiology; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Glycopeptides; Guanidines; In Vitro Techniques; Losartan; Muscle Contraction; Papillary Muscles; Patch-Clamp Techniques; Peptides, Cyclic; Sodium-Calcium Exchanger; Sulfones; Thiourea

1. Endothelin-1(1-31) (ET-1(1-31); 0.25 to 4 nmol kg(-1); i.v.) induced, in the guinea-pig, graded increases in MAP and an indomethacin-sensitive enhancement of pulmonary insufflation pressure (PIP). At all doses, ET-1(1-31) induced a monophasic pressor response, except at 4 nmol kg(-1), which caused a rapid and transient response (first phase: over first 10 min after injection) followed by a more slowly-developing and sustained (second phase: between 10 and 45 min after injection) increase in MAP. ET-1(1-31) was 4 to 10 fold less potent than ET-1 on PIP responses. 2. Phosphoramidon (5 and 10 mg kg(-1)) reduced both pressor and PIP effects of ET-1(1-31). Thiorphan (0.25 and 2.5 mg kg(-1)) did not affect the pressor responses to ET-1(1-31) although its PIP effects were markedly reduced by the NEP inhibitor. A selective endothelin-converting enzyme (ECE) inhibitor, CGS 35066 (1 mg kg(-1)), significantly reduced the second phase pressor response and increase in PIP triggered by ET-1(1-31). 3. The second (but not the first) pressor phase of ET-1(1-31) (4 nmol kg(-1)) was markedly reduced by BQ-123 (selective ET(A) antagonist), whereas the increase of PIP was significantly reduced by BQ-788 (selective ET(B) antagonist). Co-administration of BQ-123 plus BQ-788 abolished ET-1(1-31)-induced increase in PIP, but blockade of the second pressor phase afforded by BQ-123 was now reversed. 4. In guinea-pig isolated perfused lungs, ET-1(1-31) (50 nM) induced the release of prostacyclin and thromboxane A(2), which was inhibited by BQ-788 (5 nM) or thiorphan (25 microM), but not BQ-123 (1 microM). 5. These results suggest that ET-1(1-31) enhances MAP. Its sustained, but not transient, pressor effects are mediated via ET(A) receptor activation. Furthermore, ET-1(1-31) increases airway resistance in vivo and triggers prostacyclin and thromboxane A(2) release from perfused lungs predominantly via ET(B) receptor activation. ET-1(1-31) failed to display any selectivity of action towards either ET(A) or ET(B) receptors in these models. 6. We suggest that, in order to raise MAP, ET-1(1-31) requires conversion to ET-1, predominantly by ECE and to a lesser extent neutral endopeptidase 24.11, whereas the reverse holds true regarding its pharmacological effects in airways.

Topics: Analysis of Variance; Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dose-Response Relationship, Drug; Eicosanoids; Endothelin-1; Endothelin-Converting Enzymes; Female; Glycopeptides; Guinea Pigs; In Vitro Techniques; Lung; Male; Metalloendopeptidases; Neprilysin; Peptide Fragments; Pulmonary Circulation; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Respiratory Function Tests

An increased level of endothelin (ET)-1 seems to be involved in the development of augmented cerebrovascular resistance in different pathological conditions, most notably vasospasm after subarachnoid hemorrhage. Therefore, interfering with the ET synthesis or ET receptor blockade may be a promising approach in the treatment of cerebral vasospasm after subarachnoid hemorrhage. Although the receptors mediating the effects of ET-1 human cerebrovasculature are well characterized, data concerning the functionally relevant ET-converting enzyme (ECE) activity are scarce.. ECE activity was determined in organ bath studies by the use of intraoperatively harvested human pial arteries. The level of ECE activity was analyzed by comparing the shift in the concentration effect curves obtained for ET-1 and its precursor, big ET-1. In addition, the presence of ECE-1alpha immunoreactivity was studied in human cerebral tissue.. ECE-1alpha immunoreactivity was found, although not consistently, in human cerebral arteries and was restricted to the endothelium. In isolated pial arterial segments, ET-1 and big ET-1 induced concentration-related contractions with mean pD(2) values of 9.25 +/- 0.34 and 7.13 +/- 0.17, respectively, yielding a 123-fold shift of big ET-1 versus mature ET-1. Preincubation with phosphoramidon (10(-4) mol/L) resulted in a small yet significant inhibition of the contraction induced by big ET-1.. The results of our study indicate the presence of functional ECE activity and ECE-1alpha immunoreactivity in human cerebral arteries. Furthermore, the data suggest the presence of ECE-like activity that differs from that of ECE-1alpha.

Topics: Adult; Arteries; Aspartic Acid Endopeptidases; Brain Neoplasms; Cerebrovascular Circulation; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Epilepsy; Female; Glycopeptides; Humans; Immunohistochemistry; In Vitro Techniques; Intraoperative Period; Isoenzymes; Male; Metalloendopeptidases; Middle Aged; Osmolar Concentration; Pia Mater; Protein Precursors; Tissue Distribution; Vasoconstriction

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High Five(TM) insect cells in suspension culture. Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from l litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM and 4.9 min(-1) respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.

Topics: Alkaline Phosphatase; Animals; Aspartic Acid Endopeptidases; Baculoviridae; Cattle; Cell Line; Chromatography; Cloning, Molecular; Edetic Acid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Activation; Enzyme Inhibitors; Genetic Vectors; Glycopeptides; Hydrogen-Ion Concentration; Insecta; Isoenzymes; Metalloendopeptidases; Protein Precursors; Sodium Chloride; Transfection

The aim of this study was to investigate whether blocking functional endothelin-converting enzyme (ECE) activity may offer a new approach to inhibit the development of cerebral vasopasm after subarachnoid hemorrhage (SAH) by preventing transformation of big Endothelin-1 (big ET-1) to vasoactive Endothelin-1 (ET-1).. In vitro, the effect of potential ECE inhibitors was determined by measurement of isometric contractions, induced by big ET-1, in isolated rat basilar arteries. Endothelium intact (E+) and de-endothelialized (E-) segments were examined after pre-incubated with the putative ECE inhibitors: Phosphoramidon (10(-4) M), Captopril (10(-3) M and 10(-4) M) and [(22)D-Val] big ET-1 (16-38) (10(-5) M and 10(-6) M).. Application of 10(-4) M Phosphoramidon resulted in a statistically significant decrease in big ET-1 induced contraction in endothelium intact (E+) and de-endothelialized (E-) segments; 10(-3) M Captopril in E- segments caused a statistically significant inhibitory effect; 10(-4) M and 10(-3) M Captopril in E+ segments showed no statistically significant effect; 10(-5) M and 10(-6) M [(22)D-Val] big ET-1 (16-38) in E- segments produced no statistically significant effect. The application of 10(-6) M [(22)D-Val] big ET-1 (16-38) in E+ segments caused increased contractions as shown by the shift to the left of the concentration-effect curve (CEC).. The present study indicates the existence of functional ECE activity in rat basilar artery, which is different in the endothelium and the smooth muscle layer. This ECE-activity could be inhibited by Captopril and Phosporamidon, suggesting a potency for prevention and therapy of cerebral vasospasm. However, the structural analogue of big ET-1, [(22)D-Val] big ET-1 (16-38), was ineffective in reducing big ET-1 induced vasoconstriction and rather increased contraction in E+ vessels. Therefore further studies of the biochemical nature of the functional relevant cerebrovascular ECE activity are required for better understanding and development of other efficient ECE inhibitors.

Topics: Animals; Aspartic Acid Endopeptidases; Basilar Artery; Captopril; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; Glycopeptides; Male; Metalloendopeptidases; Protein Precursors; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Vascular Resistance; Vasospasm, Intracranial

Endothelin (ET)-1[1-21] stimulates steroid secretion and zona glomerulosa growth and is expressed in the human and rat adrenal cortex together with its receptor subtypes A and B (ETA and ETB). Although ET-1[1-21] is generated from bigET-1 by an ET-converting enzyme (ECE-1), there is evidence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, the role of which in adrenal pathophysiology is largely unknown. Gene expression and immunohistochemical studies allowed localization of chymase in the normal human adrenal cortex. Sizable amounts, not only of ET-1[1-21] but also of ET-1[1-31], were found in the adrenal vein plasma of three patients. ET-1[1-21] and ET-1[1-31] elicited a clear-cut secretory response by dispersed human adrenocortical cells, ET-1[1-31] being significantly less potent than ET-1[1-21]. The secretagogue effect of ET-1[1-31] was abolished by the ETA receptor antagonist BQ-123 and was unaffected by the ETB receptor antagonist BQ-788. Because, in humans, the secretagogue effect of ET-1[1-21] involves both ETA and ETB receptors, the weaker action of ET-1[1-31] could be attributable to a selective ETA receptor activation. Two lines of evidence support this contention: 1) ET-1[1-31] was more effective than ET-1[1-21] in stimulating ETA-mediated cell proliferation of human adrenocortical cells cultured in vitro; and 2) autoradiography showed that a) ET-1[1-31] displaced in vitro [(125)I]ET-1[1-21] binding to the ETA, but not ETB receptors, in human internal thoracic artery rings; and b) BQ-123, but not BQ-788, eliminated [(125)I]ET-1[1-31] binding in the rat adrenal cortex.

Topics: Adrenal Cortex; Aldosterone; Autoradiography; Cell Division; Cells, Cultured; Chymases; Endothelin-1; Endothelins; Glycopeptides; Humans; Hydrocortisone; Immunohistochemistry; In Vitro Techniques; Muscle, Smooth, Vascular; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine Endopeptidases

In animal models, endothelin-1 (ET-1) blockade attenuates transplant vasculopathy and chronic allograft dysfunction even in the absence of cyclosporine (CsA). As CsA has side effects and ET-1 antagonism alone has significant benefits, we postulated that allograft survival could be significantly improved by combining an endothelin-converting enzyme inhibitor with low-dose CsA.. Survival of Lewis to Fisher 344 rat heterotopic cardiac allografts was determined in untreated animals and compared with those treated with high-dose CsA (62 mg/kg i.m. on day 2), low-dose CsA (25 mg/kg), an endothelin-converting enzyme inhibitor, phosphoramidon (PA, 10 mg/kg/day), or low-dose CsA + PA.. Untreated allografts had a median survival of 16 days compared with 20 days for low-dose CsA. Grafts treated with PA survived for 28 days, and combination of PA and low-dose CsA improved median survival to 47 days (P<0.01). Median survival with combination therapy was similar to that for high-dose CsA (42 days). To explore mechanisms underlying the benefits of combination therapy, cardiac allografts treated as above (n=4 each group) were explanted at 20 d and analyzed for parenchymal rejection, neointimal vasculopathy, myocardial fibrosis, and macrophage infiltration. Low-dose CsA alone but not PA improved parenchymal rejection; in contrast, PA alone but not low-dose CsA improved vasculopathy. Both parenchymal rejection and vasculopathy were improved by combination therapy with low-dose CsA and PA. Unlike CsA, inhibition of ET-1 biosynthesis significantly reduced myocardial fibrosis in allografts.. These results suggest that the combination of low-dose CsA and endothelin-converting enzyme inhibition may prove useful to improve long-term graft survival while minimizing potential side effects of CsA.

Topics: Animals; Aspartic Acid Endopeptidases; Cyclosporine; Endothelin-1; Endothelin-Converting Enzymes; Fibrosis; Glycopeptides; Graft Rejection; Graft Survival; Heart Transplantation; Immunosuppressive Agents; Male; Metalloendopeptidases; Myocardium; Rats; Rats, Inbred F344; Rats, Inbred Lew; Transplantation, Homologous

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of Rho kinase. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Y-27632, a Rho kinase inhibitor, relaxed the spasm. However, relaxations to Y-27632 were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that, at least in the rabbit double subarachnoid hemorrhage model, vasospasm does not involve the functional upregulation of Rho kinase.

Topics: Amides; Animals; Basilar Artery; Endothelin-1; Glycopeptides; Intracellular Signaling Peptides and Proteins; Male; Muscle Relaxants, Central; Protease Inhibitors; Protein Serine-Threonine Kinases; Pyridines; Rabbits; rho-Associated Kinases; Subarachnoid Hemorrhage; Vasoconstriction; Vasospasm, Intracranial

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glycopeptides; Losartan; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Pepstatins; Peptides, Cyclic; Protease Inhibitors; Protein Precursors; Rats; Rats, Wistar; Renin

The enzymatic activity of endothelin-converting enzymes (ECE) was altered to determine the potential effect of endothelins (ET) on cerebral blood flow measured by laser Doppler flowmetry (CBF(LDF)) and the resultant neural damage of rats, made hypoxic via breathing 12% O(2) for 35 min. Intrastriatal administration of phosphoramidon (PRN, 5 microM), a dual inhibitor of ECE and neutral endopeptidase (NEP), significantly increased infarct volume to hypoxia with a significant attenuation of CBF(LDF). However, intrastriatal thiorphan (TRN, 5 microM), an inhibitor of NEP, had no effect on the CBF(LDF) responses or infarct volume induced by the hypoxic challenge. These findings showed that inhibition of ECE by PRN interfered with the vasodilator activity of ET to the hypoxic response that increased neural damage, thus suggesting that PRN-sensitive ECE is functionally active in the modulation of cerebral blood flow in rats undergoing hypoxia.

Topics: Animals; Aspartic Acid Endopeptidases; Brain; Cerebrovascular Circulation; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; Hypoxia, Brain; Laser-Doppler Flowmetry; Metalloendopeptidases; Neurons; Protease Inhibitors; Rats; Rats, Long-Evans; Vasodilation

Topics: Adrenomedullin; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Coronary Circulation; Endothelin-1; Glycopeptides; Humans; Lung; Peptides; Rats; Sepsis

Endothelin converting enzyme-1 (ECE-1) is a type II integral membrane protein and a zinc metalloendopeptidase. ECE-1 generates endothelin-1 (ET-1), the most potent vasoconstrictor yet discovered, by specific proteolytic processing of a precursor peptide, big ET-1. An insect cell expression system, which generates up to 4.3 mg of a secreted, soluble form of ECE-1 (solECE-1) per liter culture medium, has been established and solECE-1 was purified to homogeneity using five chromatographic steps. SolECE-1 expressed in insect cells could be suitable for X-ray structure determination as it is much less glycosylated than solECE-1 from mammalian cells. SolECE-1 from both sources, nonetheless, has comparable enzymatic properties. Despite apparent structural similarities, ECE-1 cleaves big ET-1 exclusively between Trp(21) and Val(22), in contrast to neprilysin, which cleaves big ET-1 at various sites. However, when linear big ET-1, in which the formation of disulfide bonds has been prevented by alkylation of the four cysteines, was used as substrate, it was cleaved by solECE-1 at multiple sites. This result indicates that secondary/tertiary structure of big ET-1 induced by disulfide bonds is essential for the specific cleavage of the Trp(21)-Val(22) bond by ECE-1. A continuous, fluorescent ECE-1 assay has been developed using a novel substrate, 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-(p-nitro-Phe(8))-Arg. This simple and rapid assay can greatly facilitate discovery of novel ECE inhibitors useful as pharmaceutical agents.

Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Baculoviridae; Bradykinin; Chromatography, High Pressure Liquid; Disulfides; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; Glycopeptides; Glycosylation; Humans; Kinetics; Metalloendopeptidases; Molecular Sequence Data; Peptide Fragments; Protein Precursors; Quinazolines; Recombinant Proteins; Spectrometry, Fluorescence; Spodoptera

All three endothelin precursor peptides, i.e. big endothelin-1 (big ET-1), big endothelin-2 (big ET-2) and big endothelin-3 (big ET-3), produced contractile responses in human isolated bronchi, demonstrating the presence of functional endothelin-converting enzyme (ECE) in this tissue. The maximal contractile responses were equal to 108.4+/-8.0% (0.1 microM big ET-1; n=4), 85.2+/-11.8% (0.1 microM big ET-2; n=7) and 43.0+/-7.2% (0.1 microM big ET-3; n=5) of the reference response to acetylcholine (1 mM). The response to big ET-1 (0.1 microM), but not endothelin-1 (ET-1, 0.1 microM), was diminished after overnight storage of the tissue at 4 degrees C, demonstrating instability of the enzyme. The responses to all three big-endothelins were significantly inhibited, by the ECE inhibitors CGS 26393 and CGS 26303, in a concentration-related manner. The responses to the mature peptides ET-1, endothelin-2 (ET-2), and endothelin-3 (ET-3) were unaffected by CGS 26393 and CGS 26303. Phosphoramidon (10 microM) also produced an inhibition of the response to big ET-1 that was equivalent to that produced by CGS 26393 (10 microM). Combination of CGS 26393 (10 microM) and phosphoramidon (10 microM) did not produce an additive inhibition. These results demonstrate the presence of functional ECE for all three big endothelins in human bronchus and inhibition of the enzyme by newly developed orally active ECE inhibitors, as well as phosphoramidon. British Journal of Pharmacology (2000) 129, 170 - 176

Topics: Aspartic Acid Endopeptidases; Bronchi; Endothelin-1; Endothelin-2; Endothelin-3; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; Glycopeptides; Humans; In Vitro Techniques; Lung; Metalloendopeptidases; Organophosphonates; Protease Inhibitors; Protein Precursors; Tetrazoles

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.

Topics: Animals; Aspartic Acid Endopeptidases; Basilar Artery; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Enzyme Inhibitors; Glycopeptides; In Vitro Techniques; Male; Metalloendopeptidases; Microscopy, Electron; Peptides, Cyclic; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Vasoconstriction; Vasodilation

The aims of this study were to define and characterize the different mechanisms and sites of clearance of plasma endothelin-1 (ET-1) and big endothelin-1 (BigET-1) and evaluate possible effects of ETA versus combined ETA and ETB receptor blockade or endothelin converting enzyme (ECE) inhibition.. Time courses and sites of clearance were evaluated in Wistar-Kyoto rats after bolus injection of radiolabelled peptides into the carotid artery before or after treatment with LU1 35252 (ETA) and bosentan (ETA and ETB) as receptor antagonists or the ECE inhibitor phosphoramidon.. The study shows that differential clearance of 125I-ET-1 and 125I-BigET-1 is mediated by distinct tissue-specific, receptor- and non-receptor-mediated mechanisms. Low levels of plasma ET-1 are rapidly cleared, mainly in the pulmonary circulation, through a low-capacity saturable ETB receptor-linked mechanism. In contrast, BigET-1 clearance is markedly slower, confined largely to liver and kidneys, is essentially non-receptor-mediated and is independent of converting enzyme activity. Acute inhibition of both ETA and ETB receptors with bosentan dramatically prolonged 125I-ET-1 plasma half-life and shifted tissue uptake from lung to liver and kidneys. Pulmonary clearance of 125I-ET-1 was decreased by chronic but not acute treatment with the specific ETA receptor antagonist LU135252. In contrast, 125I-Big-ET-1 clearance and tissue uptake were essentially unchanged by all treatments.. Plasma levels and clearance studies on ET-1 and BigET-1 may provide differential information regarding pathological changes in their separate uptake mechanisms. Such data could have diagnostic or prognostic value in pulmonary, hepatic and renal pathophysiology or future therapeutic monitoring of treatment efficacy following administration of selective receptor antagonists.

Topics: Administration, Oral; Animals; Bosentan; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glycopeptides; Injections, Intravenous; Male; Phenylpropionates; Protease Inhibitors; Protein Precursors; Pyrimidines; Rats; Rats, Inbred WKY; Receptor, Endothelin A; Receptors, Endothelin; Sulfonamides; Time Factors; Tissue Distribution

We investigated the role of endogenous endothelins in catecholamine secretion in response to transmural electrical stimulation in the retrogradely perfused rat adrenal gland. (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-++ +methy l-pentanoyl]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-+ ++pyridyl) propionic acid (FR139317; 0.03-3 microM), an endothelin ET(A) receptor antagonist, inhibited the electrical stimulation-induced epinephrine and norepinephrine output. Neither N-cis-2, 6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1- methoxycarbonyl tryptophanyl-D-norleucine (BQ-788; 0.03-3 microM), an endothelin ET(B) receptor antagonist, nor phosphoramidon (1-100 mM), an endothelin-converting enzyme inhibitor, affected the catecholamine output responses. However, the inhibition by FR139317 of the catecholamine output responses was abolished by pretreatment with phosphoramidon (100 mM) or BQ-788 (3 microM). These results indicate that activation of endothelin ET(B) receptors by endogenous endothelins inhibits the catecholamine output responses under the condition in which endothelin ET(A) receptors are blocked. Exogenous endothelin-1 (1-100 nM) did not affect the catecholamine output responses, but it inhibited the responses under treatment with phosphoramidon and FR139317. Activation of endothelin ET(A) receptors may interfere with the endothelin ET(B) receptor-mediated inhibitory action on the neuronally evoked secretion of adrenal catecholamines.

Topics: Adrenal Glands; Animals; Azepines; Catecholamines; Dose-Response Relationship, Drug; Electric Stimulation; Endothelin-1; Endothelins; Epinephrine; Glycopeptides; In Vitro Techniques; Indoles; Male; Norepinephrine; Oligopeptides; Piperidines; Rats; Rats, Wistar

We describe the pharmacological characteristics of SM-19712 (4-chloro-N-[[(4-cyano-3-methyl-1-phenyl-1H-pyrazol-5-yl)amino]carbonyl] benzenesulfonamide, monosodium salt). SM-19712 inhibited endothelin converting enzyme (ECE) solubilized from rat lung microsomes with an IC50 value of 42 nM and, at 10 - 100 microM, had no effect on other metalloproteases such as neutral endopeptidase 24.11 and angiotensin converting enzyme, showing a high specificity for ECE. In cultured porcine aortic endothelial cells, SM-19712 at 1 - 100 microM concentration-dependently inhibited the endogenous conversion of big endothelin-1 (ET-1) to ET-1 with an IC50 value of 31 microM. In anesthetized rats, either intravenous (1-30 mg/kg) or oral (10-30 mg/kg) administration of SM-19712 dose-dependently suppressed the pressor responses induced by big ET-1. In acute myocardial infarction of rabbits subjected to coronary occlusion and reperfusion, SM-19712 reduced the infarct size, the increase in serum concentration of ET-1 and the serum activity of creatinine phosphokinase. The present study demonstrates that SM-19712 is a structurally novel, nonpeptide, potent and selective inhibitor of ECE, and SM-19712 is a valuable new tool for elucidating the pathophysiological role of ECE.

Topics: Acute Disease; Animals; Aspartic Acid Endopeptidases; Disease Models, Animal; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Lung; Male; Metalloendopeptidases; Myocardial Infarction; Pressoreceptors; Rabbits; Rats; Rats, Sprague-Dawley; Substrate Specificity; Sulfonamides; Sulfonylurea Compounds; Swine

Effects of SM-19712 (4-chloro-N-[[(4-cyano-3-methyl- 1-1-phenyl- 1H-pyrazol-5-yl)amino]carbonyl] benzenesulfonamide, monosodium salt), a novel endothelin converting enzyme (ECE) inhibitor, on ischemic acute renal failure (ARF) in rats were examined in comparison with those of phosphoramidon, a conventional ECE inhibitor. ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function in ARF rats markedly decreased at 24 h after reperfusion. Intravenous bolus injection of SM-19712 (3, 10, 30 mg/kg) prior to the occlusion attenuated dose-dependently the ischemia/reperfusion-induced renal dysfunction. Histopathological examination of the kidney of ARF rats revealed severe renal damages such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion, all of which were dose-dependently attenuated by SM-19712. Protective effects of phosphoramidon (10 mg/kg) on ARF-induced functional and tissue damages were less potent than that of the same dose of SM-19712. Endothelin-1 (ET-1) content in the kidney after the ischemia/reperfusion was significantly increased, being the maximum level at 6 h after reperfusion, and this elevation was completely suppressed by the higher dose of SM-19712. Our findings support the view that renal ET-1 plays an important role in the development of ischemia/reperfusion-induced renal injury. SM-19712 may be useful in the treatment of ischemic ARF.

Topics: Acute Kidney Injury; Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Ischemia; Kidney Function Tests; Male; Metalloendopeptidases; Protective Agents; Rats; Rats, Sprague-Dawley; Sulfonamides; Sulfonylurea Compounds

In the skin, keratinocytes synthesize and secrete endothelin-(ET-1), a potent vasoconstrictor peptide which acts also as a growth factor for most skin cells. The aim of the study was to test the effects of UVA1 and the associations UVA1/B on the expression of ET-1 in normal human keratinocytes and to determine whether exogenously added prostaglandin E2 (PGE2) regulated ET-1 expression. As ET-1 is susceptible to degradation, we also evaluated whether ET-1 secretion was modulated by peptidase inhibitors. Our results showed that UVA1 (365 nm) did not modify the levels of preproET-1 mRNA and protein. Moreover, the associations UVA1+UVB or UVB+UVA1 down-regulated the overexpression of secreted ET-1 induced by UVB alone. PGE2 at 10(-5) M reduced the expression of ET-1 at the mRNA and protein levels but did not exert any significant modification at lower concentrations from 10(-10) to 10(6) M. Phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, drastically decreased the amount of ET-1 accumulating in the culture medium in basal conditions or after UVB irradiation. Conversely, thiorphan, a specific inhibitor of neutral endopeptidase (NEP), rather increased the levels of ET-1 secretion mainly after UVB irradiation. Taken together, the results showed that normal human keratinocytes secrete and partly degrade ET-1 through ECE and NEP pathways and pointed out a differential regulation of ET-1 by UVB and UVA1 radiations without any noticeable role for PGE2.

Topics: Cells, Cultured; Dinoprostone; Endothelin-1; Endothelins; Gene Expression Regulation; Glycopeptides; Humans; Infant, Newborn; Keratinocytes; Male; Polymerase Chain Reaction; Protease Inhibitors; Protein Precursors; Skin; Thiorphan; Transcription, Genetic; Ultraviolet Rays

This study was designed to characterize the endothelin pathway in an immortalized human adenocarcinoma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC).. The release of ET-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin isoforms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-1a, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody.. Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in both cell lines phosphoramidon produced a concentration-dependent inhibition of ET-1 release and an enhanced accumulation of big-ET-1. Both HUVEC and A549 cells express the mRNAs for ppET-1, ET-A, and ET-B receptor subtypes and ECE-1 (isoforms ECE-1b, c and/or d). In addition, in HUVEC the mRNAs for ppET-2 and for the isoform ECE-1a were also detected. In A549 cells, ECE-1 had a preferential subcellular localization in the membrane fraction but was not detected in the cytosol.. Both A549 and HUVEC produce and release endothelin-1 through a specific enzymatic pathway, whether or not ECE-1 is the only enzyme involved remains to be determined. A549 might be used as a screening assay for drug discovery such as for inhibitors of endothelin-1 release.

Topics: Adenocarcinoma; Aspartic Acid Endopeptidases; Cells, Cultured; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Lung Neoplasms; Metalloendopeptidases; Protein Precursors; RNA, Messenger; Tumor Cells, Cultured; Umbilical Veins

The aim of this work was to investigate the influence of endothelin on myocardial ischemia and reperfusion in anaesthetized dogs. Animals were submitted to left thoracotomy and 120 min of left anterior descending coronary occlusion, followed by 180 min of reperfusion. Arterial blood pressure and electrocardiogram (ECG) were recorded in order to analyze heart rate (HR)-pressure product and production of ectopic beats. Infarcted areas were identified by a macroscopic staining method and infarct size was expressed as percentage of risk zone. To inhibit the effects of endothelin in a group of animals, we administered intravenously an endothelin synthesis inhibitor (phosphoramidon) and in another group, an endothelin-1 A receptor blocker (BQ-123). Phosphoramidon decreased the HR-pressure product during reperfusion period, and both, phosphoramidon and BQ-123 decreased infarct size by 40% and the number of ventricular ectopic beats by 88% and 68%, respectively, as compared to the saline treated dogs. In conclusion, endothelin seems to play a deleterious role on the myocardium submitted to ischemia and reperfusion.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Coronary Disease; Dogs; Drug Therapy, Combination; Endothelin Receptor Antagonists; Endothelin-1; Glycopeptides; Injections, Intravenous; Myocardial Infarction; Myocardial Reperfusion; Peptides, Cyclic; Receptor, Endothelin A; Receptors, Endothelin; Ventricular Fibrillation; Ventricular Premature Complexes

We have previously observed that intracoronary administration of the alpha1-adrenergic agonist phenylephrine (PE) over a period of minutes induced both an immediate and long-lasting (2 h) vasoconstriction of epicardial coronary arterioles. Because it is unlikely that alpha1-adrenergic constriction would persist for hours after removal of the agonist, this observation supports the view that another constrictor(s) is released during alpha1-adrenergic activation and induces the prolonged vasoconstriction. Therefore, we hypothesized that the prolonged microvascular constriction after PE is due to the production of endothelin (ET). We focused on ET not only because this peptide produces potent vasoconstriction but also because its vasoconstrictor action is characterized by a long duration. To test this hypothesis, the diameters of coronary arterioles (<222 micrometers) in the beating heart of pentobarbital-anesthetized dogs with stroboscopic intravital microscopy were measured during a 15-min intracoronary infusion of PE (1 microgram. kg-1 . min-1) and at 15-min intervals for a total of 120 min. All experiments were performed in the presence of beta-adrenergic blockade with propranolol. At 120 min, arterioles in the PE group were constricted (-23 +/- 9% change in diameter vs. baseline). Pretreatment with the ET-converting enzyme inhibitor phosphoramidon or the ETA-receptor antagonist FR-139317 prevented the PE-induced constriction at 120 min (-1 +/- 3 and -6 +/- 3%, respectively, P < 0.01 vs. PE). Pretreatment with the selective alpha1-adrenergic antagonist prazosin (Prz) also prevented the sustained constriction (0 +/- 2%, P < 0.01 vs. PE) but Prz given 60 min after PE infusion did not (-13 +/- 3%). In the aggregate, these results show that vasoconstriction of epicardial coronary arterioles via alpha1-adrenergic activation is blocked by an ET antagonist and an inhibitor of its production. From these data, we conclude that alpha1-adrenergic activation promotes the production and/or release of ET, which produces or facilitates microvascular constriction of epicardial canine coronary arterioles.

Topics: Adrenergic alpha-Agonists; Adrenergic beta-Antagonists; Animals; Arterioles; Azepines; Coronary Vessels; Dogs; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glycopeptides; Hemodynamics; Indoles; Phenylephrine; Propranolol; Receptor, Endothelin A; Vasoconstriction

Changes in the immunoreactive ET-1 levels during the anaphylactic reaction of airway tissue from ovalbumin-sensitized guinea pigs were investigated. ET-1-immunoreactivity (ET-IR) was detected in the epithelial and smooth muscle layers of tracheal sections from normal guinea pigs and it was enhanced slightly by phosphoramidon (1 microM) treatment. The ET-IR level of the epithelial layer of ovalbumin-treated tissue from actively sensitized animals was slightly higher than that from normal animals, but it was enhanced markedly by phosphoramidon (1 microM) treatment. Furthermore, the mean ET-IR level of homogenates of antigen-treated tracheal tissues from sensitized guinea pigs (22.8 +/- 1.55 fmol mg-1 protein, n = 5) was significantly higher than the corresponding normal level (12.3 +/- 1.21 fmol mg-1 protein, n = 5). These results suggest that increased epithelial airway ET-1 levels contribute to the anaphylactic reaction of guinea pig airways.

Topics: Anaphylaxis; Animals; Antigens; Endothelin-1; Epithelium; Glycopeptides; Guinea Pigs; Immunohistochemistry; Male; Muscle, Smooth; Ovalbumin; Trachea

Endothelin-1 (ET-1) is the predominant endothelin isopeptide generated by the vascular wall and therefore appears to be the most important peptide involved in regulation of cardiovascular events. Many pathologic conditions are associated with elevations of ET-1 in the blood vessel wall. Because these conditions are often cytokine driven, we examined the effects of a mixture of cytokines on ET-1 production in human vascular smooth muscle cells (VSMCs) derived from internal mammary artery and saphenous vein (SV). Incubation of IMA and SV VSMCs with tumor necrosis factor-alpha (10 ng/ml) and interferon-gamma (1000 U/ml) in combination for up to 48 h markedly elevated the expression of mRNA for prepro-ET-1 and the release of ET-1 into the culture medium. This cytokine-stimulated release of ET-1 was inhibited by a series of dual endothelin-converting enzyme (ECE)/neutral endopeptidase inhibitors, phosphoramidon, CGS 26303, and CGS 26393, with an accompanying increase in big ET-1 release but with no effect on expression of mRNA for prepro-ET-1. These same compounds were 10 times more potent at inhibiting the conversion of exogenously applied big ET-1 to ET-1. ECE-1b/c mRNA is present in SV VSMCs, however no ECE-1a is present in these cells. Thus VSMCs most probably contain, like endothelial cells, an intracellular ECE responsible for the endogenous synthesis of ET-1. Under the influence of pro-inflammatory mediators the vascular smooth muscle can therefore become an important site of ET-1 production, as has already been established for the dilator mediators nitric oxide, prostaglandin I2, and prostaglandin E2.

Topics: Aspartic Acid Endopeptidases; Cells, Cultured; Cytokines; Drug Interactions; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; Humans; Interferon-gamma; Mammary Arteries; Metalloendopeptidases; Muscle, Smooth, Vascular; Organophosphonates; Phenylalanine; Protease Inhibitors; RNA, Messenger; Saphenous Vein; Tetrazoles; Tumor Necrosis Factor-alpha

The current studies were undertaken to investigate the role of endothelin-1 (ET-1) and its receptors in contractions of isolated pulmonary vessels of the pig induced by diaspirin cross-linked hemoglobin (DCLHb). Second-order pulmonary arteries (PAs) and veins (PVs) were isolated from pigs, cut into rings (4 to 5 mm), and mounted at optimal passive tension in 37 degrees C Krebs-filled tissue baths bubbled with 95% O2/5% CO2. Isometric tension was recorded continuously. In paired rings, concentration responses to ET-1 (10(-10) to 10(-7) mol/L), DCLHb (10(-9) to 3x10(-6) mol/L), and N-nitro-L-arginine (LNA) (10(-6) to 5x10(-5) mol/L) in the presence and absence of the ET(A) receptor antagonist BQ123 (3x10(-5) mol/L) were determined. PVs and PAs with intact endothelium and rings from which the endothelium was removed (denuded) were pretreated with the ET(B) receptor antagonist BQ788 to determine the contribution of ET(B) receptors to ET-1, DCLHb, and LNA responses. ET-1, DCLHb, and LNA caused concentration-dependent increases in tension in all vessels. In the presence of BQ123, the 50% effective concentration (EC50) of ET-1 was significantly increased (by 5-fold to 10-fold) in all vessels. DCLHb concentration responses were significantly attenuated-in the PVs by 45% and in the PAs by 79%-during treatment with BQ123. BQ123 attenuated LNA responses in PVs by 35% and in PAs by 87%. Treatment with BQ788 had no effect on endothelium-intact PVs or PAs but significantly increased ET-1 EC50 (log of the molar concentration) from -9.0+/-0.22 to -7.8+/-0.05 in denuded PAs. The ET-1 EC50 was significantly decreased in denuded PAs (-9.0+/-0.22) as compared with responses in endothelium-intact PAs (-8.1+/-0.18). DCLHb concentration responses were attenuated by 71% and LNA responses by 80% during antagonism with BQ788 in the intact PAs only. These data demonstrate that ET-1 plays a role in DCLHb-induced contractions in the PA and PV. The contributions of ET are mediated by both ET(A) and ET(B) receptors in the PA but only by ET(A) receptors in the PV. These results suggest that the vasoconstrictor actions of DCLHb, which have previously been shown to depend on its interference with endothelium-generated NO, may also involve ET. This may reflect the importance of the interaction of these two endothelium-generated physiologic antagonists in the pulmonary circulation.

Topics: Animals; Aspirin; Endothelin Receptor Antagonists; Endothelin-1; Glycopeptides; Hemoglobins; Isometric Contraction; Muscle, Smooth, Vascular; Nitroarginine; Oligopeptides; Piperidines; Pulmonary Artery; Pulmonary Veins; Receptor, Endothelin A; Receptor, Endothelin B; Swine; Vasoconstrictor Agents

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of heart disease in Latin America. T. cruzi-induced microvascular compromise, in turn, is thought to play a major role in chagasic heart disease. Previous in vitro studies have implicated endothelin-1 (ET-1) as a potentially important vasomodulator present in increased levels in the supernatant of T. cruzi infected cultured human umbilical vein endothelial cells (HUVEC). Thus, the goal of the present investigation was to further evaluate the potentially important contribution of ET-1 to T. cruzi-induced alterations in vascular tone in vitro. Bioassay studies once again documented that exposure of isolated rat aortic rings to infected HUVEC supernatants elicited contractile responses whose steady-state magnitude was significantly greater than contractile responses elicited by exposure of aortic rings to uninfected HUVEC supernatants. Furthermore, the increased aortic contractility was significantly attenuated by the presence of the ET(A) subtype selective antagonists BMS-182,874 or BQ-123. Additionally, incubation of HUVEC with either verapamil or phosphoramidon prior to infection was also associated with reduced aortic contractility, upon exposure to the supernatant. Phosphoramidon, but not verapamil, produced a significant decrease in the measured ET-1 levels in the HUVEC supernatant. Consistent with the bioassay results, preincubation of Fura-2-loaded cultured rat aortic vascular smooth muscle cells with verapamil resulted in a near complete ablation of ET-1-induced transmembrane Ca2+ flux. Taken together, these data are consistent with the hypothesis that ET-1-induced vasoconstriction may play an important modulatory role in the vascular compromise characteristic of T. cruzi infection.

Topics: Animals; Aorta; Calcium; Cells, Cultured; Chagas Cardiomyopathy; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Glycopeptides; Humans; Intracellular Fluid; Mice; Muscle Contraction; Muscle, Smooth, Vascular; Peptides, Cyclic; Protease Inhibitors; Rats; Receptor, Endothelin A; Trypanosoma cruzi; Vasoconstriction; Vasodilator Agents; Verapamil

Transplant vasculopathy in kidney and heart allografts is associated with marked elevation of endothelin-1 (ET-1), but a role for ET-1 in the pathogenesis of transplant vasculopathy and chronic rejection has not been established. We, therefore, tested whether inhibition of ET-1-converting enzyme by phosphoramidon (PA) would attenuate rejection in a rat model of chronic cardiac allograft rejection (Lewis [LEW] to F344).. Donor LEW rats were pretreated 24 hr before transplantation with a bolus injection of vehicle (water) or PA. Twenty- four hour after transplantation, water or PA was continuously administered through an osmotic mini-pump. Plasma ET-1 levels in Fisher 344 (F344) recipients were 0.8+/-0.1 pg/ml in water-treated rats and 0.2+/-0.2 pg/ml (P<0.01) in PA-treated rats, demonstrating that the PA treatment protocol effectively lowered ET-1 biosynthesis.. LEW cardiac allografts treated with water survived (i.e., palpable heart beat) for 16.0+/-0.5 days (n=6). Inhibition of ET-1 secretion by PA improved allograft survival to 28.8+/-3.3 days (P<0.01, n=8). An analysis of cardiac arteries demonstrated that PA treatment attenuated transplant vasculopathy. A morphometric scale of neointima formation (0-5) was 1.4+/-0.2 and 3.6+/-0.2 in PA- or water-treated rats, respectively (P<0.01). The percent of luminal occlusion, as measured by microscopic image analysis, was 19+/-6% in PA-treated animals and 38+/-6% (P<0.01) in animals treated with water. PA treatment also reduced infiltration of ED-1-positive monocytes/macrophages into the vascular neointima.. We conclude that, even in the absence of concomitant immunosuppression, inhibition of ET-1 biosynthesis significantly attenuates transplant vasculopathy and improves survival of LEW to F344 cardiac allografts.

Topics: Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; Graft Rejection; Graft Survival; Heart Transplantation; Macrophages; Metalloendopeptidases; Protease Inhibitors; Rats; Rats, Inbred F344; Rats, Inbred Lew; Transplantation, Homologous; Vascular Diseases

The effects of endothelin (ET)-1(1-31) and ET-2(1-31), human chymase products of the corresponding big ETs, on the intracellular free Ca2+ concentration ([Ca2+]i) and [125I]-ET-1 binding were investigated using human cultured bronchial smooth muscle cells (BSMC). ET-1(1-31) (10(-8)M - 3 x 10(-7)M) and ET-2(1-31) (3 x 10(-8)M - 3 x 10(-6) M) caused an increase in [Ca2+]i in a concentration-dependent manner. Big ET-1 (3 x 10(-8)M - 10(-6)M) also caused this increase, but not big ET-2 at concentrations up to 10(-6)M. The [Ca2+]i increase induced by ET-1 was inhibited by both BQ123, an ET(A)-receptor antagonist, and BQ788, an ET(B)-receptor antagonist, whereas that induced by ET-3 was inhibited by BQ788 but not by BQ123. Increases in [Ca2+]i caused by ET-1(1-31), big ET-1 and ET-2(1-31) were completely inhibited by 10(-4)M phosphoramidon, a dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor, and 10(-5)M thiorphan, a NEP inhibitor. Scatchard plot analyses of the saturation curves of [125I]-ET-1 and [125I]-ET-3 showed that both ET(A)- and ET(B)- receptors at the ratio of 4:1 were expressed on BSMC. ET-1(1-31), big ET-1 and ET-2(1-31) inhibited [125I]-ET-1 binding in a concentration-dependent manner, and these effects were attenuated by treatment with thiorphan. On the other hand, big ET-2 slightly inhibited the binding at a high concentration and this was not affected by thiorphan. These results suggest that ET-1(1-31), big ET-1 and ET-2(1-31) cause an increase in [Ca2+]i by being converted into the corresponding ET-1 and ET-2 by NEP, but this did not occur with big ET-2 in human BSMC. ET-2(1-31), produced by human chymase from big ET-2 might be important for the generation of ET-2 in human bronchial tissue.

Topics: Adult; Binding, Competitive; Bronchi; Calcium; Cells, Cultured; Endothelin-1; Endothelin-3; Endothelins; Glycopeptides; Humans; Iodine Radioisotopes; Male; Muscle, Smooth; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Protease Inhibitors; Radioligand Assay; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin

We have determined the nucleotide sequence of the clone pAL2 obtained from Porphyromonas gingivalis 381 in the previous study [Ansai et al. (1995) Microbiology 141, 2047-20521. The DNA sequence analysis of this fragment revealed one complete ORF and one incomplete ORF. The ORF encoded a protein (PgPepO) of 690 amino acids with a calculated molecular weight of 78796. The deduced amino acid sequence exhibited a significant homology with human endothelin-converting enzyme (ECE)-1. Recombinant PgPepO was purified to homogeneity and characterized. The purified enzyme was strongly inhibited by phosphoramidon, and converted big endothelin-1 to endothelin-1. Furthermore, the purified PgPepO strongly cross-reacted with a monoclonal antibody against rat ECE-1. These results indicate that PgPepO has striking similarity to mammalian ECE in structure and function.

Topics: Amino Acid Sequence; Aspartic Acid Endopeptidases; Bacterial Proteins; Base Sequence; Blotting, Southern; Cloning, Molecular; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; Genes, Bacterial; Glycopeptides; Humans; Kinetics; Metalloendopeptidases; Molecular Sequence Data; Porphyromonas gingivalis; Protein Precursors; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity

We found that human chymase selectively produces 31-amino-acid length endothelins (1-31) (ETs(1-31)). We investigated the effect of synthetic ET-1(1-31) on intracellular free Ca2+ concentration ([Ca2+]i) in cultured human mesangial cells. ET-1(1-31) increased [Ca2+]i in a concentration-dependent manner to a similar extent as ET-1. The ET-1 (1-31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon. It was inhibited by BQ123, but not by BQ788. These results suggest that ET-1(1-31) by itself exhibits bioactive properties probably through endothelin ET(A) or ET(A)-like receptors. Since human chymase has been reported to exist in the kidney, ET-1(1-31) may be a candidate substance for mesangium-relevant diseases.

Topics: Aspartic Acid Endopeptidases; Calcium; Cells, Cultured; Chymases; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glomerular Mesangium; Glycopeptides; Humans; Intracellular Fluid; Metalloendopeptidases; Microscopy, Confocal; Peptide Fragments; Peptides, Cyclic; Protease Inhibitors; Serine Endopeptidases; Thapsigargin

Vasodilation of resistance vessels ensues in response to increased perfusion flow to maintain tissue perfusion. The flow-induced vasodilation is mainly dependent on nitric oxide (NO), which also regulates vascular responsiveness to vasoconstrictors. Besides NO, however; high flow increases endothelin-1 (ET-1) production from endothelial cells. It is likely, therefore, that the interaction between NO and ET-1 may play a critical role in the control of arterial vascular tone under high perfusion flow. In this study, the vascular responsiveness (VR) to high flow rate and the role of ET-1 released by vascular smooth muscle cells (VSMC) were evaluated in isolated and in vitro-perfused mesenteric arteries (MA). MA were perfused at constant (3.5 mL/min; CPF) and increased flow rate (4.5, 5.5, 6.5 mL/min; IPF). VR was evaluated by infusing norepinephrine (NE; 5 micromol/L) and potassium chloride (KCl; 80 mmol/L). Mesenteric vascular resistance (MVR), ET-1, and cGMP release were measured under different flow rates. The role of endothelium-derived ET-1 was evaluated by perfusing MA with phosphoramidon (endothelin converting enzyme inhibitor), whereas the role of other endothelium-derived vasoactive substances was excluded by measuring VR in MA without endothelium. Finally, ETA and ETB receptor antagonists were perfused in disendothelized MA. In the IPF group of intact MA, MVR dropped (P<.05) and both ET-1 and cGMP increased in the perfusate (P<.05). VR was enhanced by high flow after NE (101+/-9 v. 56+/-12 mm Hg in CPF, P<.005) and KCl (119+/-12 v. 51+/-10 mm Hg in CPF, P<.005) and it was unaffected by either phosphoramidon or endothelium removal. On the contrary, BQ-610 abolished the flow-dependent increase in VR. No further additive effect was achieved with BQ-788. In conclusion, in MA, high flow reduces MVR and concurrently enhances VR, likely through VSMC-derived ET-1.

Topics: Animals; Antihypertensive Agents; Aspartic Acid Endopeptidases; Blood Flow Velocity; Cyclic GMP; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; In Vitro Techniques; Mesenteric Arteries; Metalloendopeptidases; Muscle, Smooth, Vascular; Norepinephrine; Perfusion; Potassium Chloride; Pressure; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Vascular Resistance; Vasoconstrictor Agents; Vasodilation

Peroxynitrite plays an important role in the pathogenesis of airway inflammation. We have already found that peroxynitrite may contribute to decreased beta(2)-adrenoceptor responses in airway smooth muscle. However, it is not known whether peroxynitrite can alter neutral endopeptidase (EC 3.4.24.11; NEP) activity in the airways. This study was designed to determine whether peroxynitrite induces airway hyperresponsiveness to substance P (SP) and endothelin-1 (ET-1) through the inactivation of airway NEP.. We examined whether the administration of S-morpholinosydnonimine (SIN-1), a compound that releases peroxynitrite, increased bronchoconstrictor responses to SP and ET-1 in anesthetized guinea pigs. In addition, we assayed NEP activity in the airways of SIN-1-exposed guinea pigs.. Though SIN-1 (10(-7) M) alone had no effect on pulmonary resistance, pretreatment with SIN-1 significantly enhanced SP- and ET-1-induced bronchoconstriction. Pretreatment with phosphoramidon, an NEP inhibitor, also enhanced SP- and ET-1-induced bronchoconstriction. However, simultaneous administration of phosphoramidon and SIN-1 had no additive effect on SP- and ET-1-induced bronchoconstriction. Peroxynitrite formation by SIN-1 was completely inhibited by N-acetylcysteine (NAC) and glutathione (GSH) in vitro, and pretreatment with NAC and GSH significantly reversed the potentiation by SIN-1 of SP-induced bronchoconstriction. In addition, the NEP activity of the trachea after SIN-1 exposure was significantly reduced compared to the level in control guinea pigs (solvent for SIN-1: 30.0+/-4.2 fmol.min(-1).mg tissue(-1); 10(-7) M SIN-1; 15.5+/-4.5 fmol.min(-1).mg tissue(-1), p<0.05).. These findings suggest that peroxynitrite induces airway hyperresponsiveness to SP and ET-1 through the inactivation of airway NEP, and that peroxynitrite is an important mediator of the alterations in airway functions.

Topics: Animals; Bronchial Hyperreactivity; Bronchoconstriction; Endothelin-1; Enzyme Activation; Glycopeptides; Guinea Pigs; Male; Molsidomine; Neprilysin; Nitrates; Oxidants; Protease Inhibitors; Respiratory System; Substance P; Trachea; Vasodilator Agents

Endothelin-1 (ET-1) is produced from inactive precursor big ET-1 by endothelin-converting enzyme-1 (ECE-1), a membrane-bound metalloprotease, structurally similar to another metalloprotease, neutral endopeptidase 24.11 (NEP). Although both phosphoramidon and thiorphan are metalloprotease inhibitors, the ECE activity is inhibited by phosphoramidon but not by thiorphan, a specific inhibitor of NEP. Therefore, to investigate whether an ECE inhibitor can prevent indomethacin-induced gastric mucosal damage in rats, we compared the effects between the two metalloprotease inhibitors on both gastric mucosal integrity and the levels of ET-1 and big ET-1 in gastric tissue. Phosphoramidon significantly decreased ET-1 levels, causing a concomitant big ET-1 increase and dose-dependently attenuated indomethacin-induced gastric mucosal damage. By contrast, thiorphan neither changed the ratio of ET-1/big ET-1 nor attenuated the damage. In conclusion, the prevention of gastric mucosal damage by an ECE inhibitor indicates that endogenous ET-1 may play an important role in the pathogenesis of indomethacin-induced gastric mucosal damage.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Gastric Mucosa; Glycopeptides; Indomethacin; Male; Metalloendopeptidases; Protease Inhibitors; Protein Precursors; Rats; Rats, Wistar; Stomach Diseases

To study the conversion of human big endothelin-1 (bigET-1) to endothelin-1 (ET-1) and to characterize contractile ET-1 receptors in human placental arteries.. BigET-1 was incubated with artery membranes and the formation of ET-1 was investigated. ET-1 and bigET-1-induced contractile responses were studied in the absence or presence of the metalloprotease inhibitor phosphoramidon, the ET(A)-receptor antagonist BQ 123, or the ETB-receptor antagonists IRL 1038 and RES 701-1.. The artery membranes hydrolysed bigET-1 to ET-1 through a partly phosphoramidon-sensitive pathway. The contractile responses to ET-1 and bigET-1 were similar, with pEC50% values of 8.1 +/- 0.2 and 7.8 +/- 0.1, respectively (NS; n = 17). Phosphoramidon decreased pEC50% for bigET-1-evoked contractions (p < 0.05; n = 8), without affecting the response to ET-1. A Schild plot of BQ 123 effects on ET-1 and bigET-1-induced contractions resulted in identical pA2 values and a slope of 0.56 +/- 0.2 and 0.47 +/- 0.01, respectively. IRL 1038 and RES 701-1 did not affect the contractile responses.. BigET-1-evoked contractions in isolated human placental arteries depend on a rapid and metalloprotease-dependent hydrolytic conversion to ET-1, which in turn causes a, mainly ETA-receptor-mediated, contraction.

Topics: Arteries; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Enzyme Inhibitors; Female; Glycopeptides; Humans; Membrane Proteins; Muscle Contraction; Muscle, Smooth, Vascular; Osmolar Concentration; Peptide Fragments; Peptides, Cyclic; Placenta; Protein Precursors; Trypsin Inhibitors; Umbilical Arteries

Myocardial ischemia plays a central role in the development of right ventricular failure after acute pulmonary embolism. This study investigates whether pulmonary mediators act specifically on coronary tone and cardiac contractile function in acute pulmonary microembolization and whether such effects are altered in the case of early systemic atherosclerosis. We employ a novel model of serial perfusion in which an isolated rabbit heart is perfused with the effluent of the same animal's isolated lung.. Controlled experiment using isolated organs.. Experimental laboratory.. Male New Zealand White rabbits (controls). Age-matched, male Watanabe rabbits (hypercholesterolemic, development of accelerated atherosclerosis).. Seven isolated control and seven isolated Watanabe hearts were perfused with the saline effluent of the same animal's isolated lung. After the assessment of the baseline data, the lungs were gradually embolized with glass beads measuring 100 microm in diameter to induce an increase in mean pulmonary arterial pressure from 6 to 8 mm Hg, at baseline, up to 25 mm Hg.. Pulmonary embolization to 25 mm Hg evoked a coronary constriction, measured as coronary flow decrease to 89 +/- 7% of the baseline value in controls. In the Watanabe group, coronary constriction was significantly enhanced, compared with controls, with coronary flow decreasing to 76 +/- 6% of the baseline value. In both groups, coronary constriction was followed by a deterioration in cardiac contractile performance. This cardiodepression was significantly deeper in Watanabe hearts with respect to both maximum ventricular pressures and maximum rates of pressure development and decline. Coronary constriction and cardiodepression were prevented by coronary infusion of the nonselective endothelin antagonist PD-145065, the endothelinA antagonists A-127722 and BQ-123, and the endothelin-converting enzyme inhibitor phosphoramidon. Concentration of big endothelin in pulmonary effluent increased from 5.6 +/- 0.3 pmol/L in controls and 5.6 +/- 0.2 pmol/L in the Watanabe group, at baseline, to 8.8 +/- 0.4 pmol/L in controls and 8.9 +/- 0.4 pmol/L in the Watanabe group, at 25 mm Hg pulmonary arterial pressure. Endothelin was not detectable at any time during the experiment in pulmonary effluent. The coronary gradient, calculated as a difference in concentration between coronary and pulmonary effluent, was negative for big endothelin and positive for endothelin in both groups.. We have demonstrated that an increase in pulmonary release of big endothelin occurs during lung embolism, which, in turn, results in coronary constriction and consequent cardiodepression. This action of big endothelin is based on its local coronary conversion into endothelin. In addition, coronary endothelial dysfunction, attributed to early systemic atherosclerosis, was shown to represent a specific risk factor in these events.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arteriosclerosis; Aspartic Acid Endopeptidases; Atrasentan; Coronary Circulation; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; In Vitro Techniques; Male; Metalloendopeptidases; Myocardial Contraction; Oligopeptides; Peptides, Cyclic; Protein Precursors; Pulmonary Embolism; Pyrrolidines; Rabbits; Thromboxane B2; Vasoconstriction

Endothelins (ET) are potent vasoactive peptides present in many ocular structures and are formed from precursor Big endothelins (Big ET-1) by the action of an endothelin-converting enzyme (ECE). ET-1 is thought to decrease intraocular pressure by contracting the ciliary muscle thus enhancing the outflow of aqueous humor through the Canal of Schlemm and trabecular meshwork. However, the mechanisms involved in the regulation of endothelin-1 (ET-1) synthesis and release in ocular tissues have not been fully characterized. In this study we examined the effect of tumor necrosis factor-alpha(TNF-alpha; 10 nm), a proinflammatory cytokine, on the cellular mechanisms leading to ET-1 synthesis and release in SV-40 transformed human ciliary non-pigmented epithelial cells (HNPE). ET-1 and Big endothelin-1 (Big ET-1) immunoreactivity was time-dependently increased following TNF-alphatreatment. Phorbol esters (PMA), activators of PKC, also raised the immunoreactive levels of ET-1 and Big ET-1 while, staurosporine, a PKC inhibitor (20 nm), decreased ET-1 levels in TNF-alpha-stimulated cells. Pre-treatment with phosphoramidon (1 micron) an ECE-inhibitor, followed by TNF-alpha stimulation, decreased ir-ET-1 levels. Cycloheximide (9 micron), a protein synthesis inhibitor, decreased TNF-alpha-stimulated levels for ir-ET-1 and ir-Big ET-1, suggesting that TNF-alpha may be directly regulating ET-1 expression at the ET-1 gene. Our data indicates that TNF-alpha regulates ET-1 levels in HNPE cells possibly by activating PKC either to stimulate protein synthesis and/or to enhance ET-1 secretion. These results suggest that ET-1 released from the ciliary body may play an important role in aqueous humor dynamics following cytokine activation.

Topics: Cells, Cultured; Ciliary Body; Cycloheximide; Endothelin-1; Epithelial Cells; Fluorescent Antibody Technique; Glycopeptides; Humans; Protease Inhibitors; Protein Kinase C; Protein Synthesis Inhibitors; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

The human megakaryoblastic cell lines HEL, MEG-01, and DAMI express preproendothelin-1 mRNA. This investigation was designed to find out whether they could also express endothelin-converting enzyme (ECE) and release mature endothelin (ET). RT-PCR applied to RNA isolated from the cell lines amplified fragments of the expected size. The amplified cDNA of MEG-01 was submitted to restriction enzymes, which generated the expected subfragments. Membrane ECE activity was phosphoramidon-sensitive, in contrast to the cytosolic activity capable of producing ET-1 from big ET-1. The three cell lines produced ir-ET in a time-dependent manner. These results show that human megakaryoblastic cell lines express functional, phosphoramidon-sensitive and insensitive ECE activity and produce mature ET.

Topics: Aspartic Acid Endopeptidases; Cell Line; DNA, Complementary; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; Humans; Megakaryocytes; Membranes; Metalloendopeptidases; Polymerase Chain Reaction; Protease Inhibitors

We have previously found that human chymase cleaves big endothelins at the Tyr31-Gly32 bond and produces 31-amino acid long endothelins-(1-31), without any further degradation products. In this study, we investigated the effect of synthetic endothelin-1-(1-31) on the intracellular free Ca2+ concentration ([Ca2+]i) in cultured human coronary artery smooth muscle cells. Endothelin-1-(1-31) increased [Ca2+]i in a concentration-dependent manner (10(-14) to 10(-10) M). This endothelin-1-(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon (N-(alpha-Rhamnopyranosyloxyhydroxyphosphinyl)-L-Leucyl-L-Tryptoph an), an inhibitor of endothelin-converting enzyme. It was, however, inhibited by 10(-10) M BQ123 (Cyclo-(-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu-)), an endothelin ET(A) receptor antagonist, but not by 10(-10) M BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-yMeLeu-D-Trp(COOM e)-D-Nle-ONa), an endothelin ET(B) receptor antagonist. These results suggest that endothelin-1-(1-31) by itself exhibits vasoactive properties probably through endothelin ET(A) receptors. Since human chymase has been reported to play a role in atherosclerosis, endothelin-1-(1-31) may be one of the candidate substances for its cause.

Topics: Calcium; Cells, Cultured; Coronary Vessels; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glycopeptides; Humans; Metalloendopeptidases; Microscopy, Confocal; Muscle, Smooth, Vascular; Peptide Fragments; Peptides, Cyclic; Protease Inhibitors

The stoichiometric binding model (Frelin C, Guedin D. Cardiovasc Res 1994;28:1613-1622) implies that most endothelin in tissues is bound onto receptors rather than in a free state. The objective of this study was to assay receptor bound endothelins in normal rat tissues.. We first defined acidic conditions that promoted a mild and reversible denaturation of ETA and ETB receptors and that allowed dissociation of bound [125I]endothelin-1. The action of an acid wash on [125I]endothelin-1 binding to cell or tissue homogenates was then investigated.. Acid washing of homogenates prepared from rat brain capillary endothelial cells that express prepro endothelin-1 mRNAs unmasked receptor sites. Acid washing of cardiac, lung, brain or liver homogenates did not increase [125I]endothelin-1 binding. An acid wash of kidney homogenates increased 2.2 fold [125I]endothelin-1 binding. Experiments using BQ-123 further indicated that the acid treatment of renal homogenates mainly unmasked ETA receptors. Masked renal ET receptors were mainly localized in the medulla. Treatment of rats with phosphoramidon decreased the density of masked ET receptors in kidney homogenates.. As much as 50% of endothelin receptors in renal tissues are masked by endogenous endothelins. Most cardiac receptors are free of bound endothelins. These suggest that endothelins act as local rather than systemic mediators.

Topics: Animals; Endothelin Receptor Antagonists; Endothelin-1; Female; Glycopeptides; Heart; Hydrogen-Ion Concentration; Iodine Radioisotopes; Kidney; Male; Myocardium; Peptides, Cyclic; Protease Inhibitors; Protein Binding; Rats; Rats, Wistar; Receptors, Endothelin

We investigated whether endogenous pulmonary big endothelin has arrhythmogenic properties under normal conditions and in heritable hyperlipidemia.. Endothelin (ET), one of the most potent vasoconstrictors, is known to induce ventricular arrhythmias. It is unclear, however, whether its precursor, big endothelin, released from the lung, contributes to arrhythmogenesis.. In a lung-heart model in which a Langendorff heart is serially perfused with the effluent from the isolated lung of the same animal, we evaluated arrhythmias in control and in Watanabe heritable hyperlipidemic (WHHL) rabbits.. In both controls (n=12) and WHHL (n=8), serial perfusion evoked a decrease in coronary flow (controls, -11+/-3%; WHHL, -25+/-6%) and a fourfold increase of ventricular extrasystoles (VES) (controls, 40.7+/-8; WHHL, 40.2+/-5 VES/40 min, p < 0.05). However, WHHL developed more and longer nonsustained ventricular tachycardias (VT) compared with controls (incidence, 1.38+/-1.1 vs. 0.33+/-0.5 VT/40 min, p < 0.05; length, 14.36+/-3.1 vs. 7.25+/-1.5 beats/VT, p < 0.05). Arrhythmias were not ischemia-induced because corresponding mechanical flow reduction had no arrhythmogenic effect (n=6 in controls and WHHL). Although vasoconstriction disappeared entirely, arrhythmias were only partly suppressed by ET(A) antagonists (BQ-123, 2 micromol/liter; A-127722, 20 micromol/liter). The ET-converting enzyme inhibitor phosphoramidon (50 micromol/liter) completely suppressed arrhythmias and vasoconstriction. The ET(B) antagonists (IRL-1038, 4 micromol/liter; IRL-1025, 5 micromol/liter) had no effect (n=6).. Endogenous pulmonary big ET produces arrhythmogenic effects that are aggravated in heritable hyperlipidemia. These effects, requiring coronary conversion of big ET into ET, are partly ET(A)-mediated and ET(B)-independent.

Topics: Animals; Aspartic Acid Endopeptidases; Cardiac Complexes, Premature; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hyperlipidemias; In Vitro Techniques; Male; Metalloendopeptidases; Myocardium; Perfusion; Protease Inhibitors; Protein Precursors; Rabbits; Tachycardia, Ventricular

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dipeptides; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; Heart Rate; Hemodynamics; Metalloendopeptidases; Protease Inhibitors; Protein Precursors; Rats; Rats, Wistar; Succinates

To examine the relationship between the profound hypotension in endotoxic shock and the dynamic changes of nitric oxide (NO) and endothelin-1 (ET-1), so as to figure out which of the NO or ET-1 was more involved in the pathogenesis of endotoxic shock. And to investigate whether an offset of their opposite vasoactive effects would occur during endotoxic shock.. 24 rabbits were anesthetized and instrumented for recording hemodynamics. Endotoxin (E. coli 026: B6, 600 micrograms/kg) was bolus injected intravenously and the animals were randomly divided into four groups. Group I was control without any more intervention, and Group II, III, IV received bolus injections of L-NMA (10 mg/kg), phosphoramidon (2 mg/kg) or dexamethasone (2 mg/kg) respectively at 30 min post-endotoxin. Plasma NO3-, ET-1 and hemodynamics were measured at regular intervals. Their relationships were compared and analysed.. Plasma ET-1 achieved its peak level at 60 min post-endotoxin, and then waned. Plasma NO3- started rising at 120 min post-endotoxin, then progressive increase continued till the last measurement at 180 min post-endotoxin. The decrease of blood pressure was significant at about 120 min post-endotoxin and further went down until death. The changes of hemodynamics and NO showed a quite close temporal correspondence between the increase of NO and the decrease of blood pressure. L-NMA and phosphoramidon obviously reduced the plasma levels of NO and ET-1 to below their respective baseline levels, and showed transient effect of increase on blood pressure. Soon afterwards, however, the status of hemodynamics was aggravated. Dexamethasone just inhibited the excessive increase of NO and ET-1 during endotoxic shock without interfering their baseline levels and showed most beneficial effects on hemodynamics.. Both NO and ET-1 increase during endotoxic shock, but only the increase of NO has a close temporal correspondence with the decrease of blood pressure. It suggests a more important role of NO in pathogenesis of endotoxic shock. The increase of NO and ET-1 is different in time-process, which indicates that an offset of their opposite vasoactive effects would not occur. Intreference against the increase of NO and ET-1 during endotoxic shock is most beneficial when their baseline levels are maintained.

Topics: Animals; Blood Pressure; Dexamethasone; Endothelin-1; Glycopeptides; Hemodynamics; Male; Nitric Oxide; omega-N-Methylarginine; Rabbits; Random Allocation; Shock, Septic

1. An obligatory step in the biosynthesis of endothelin-1 (ET-1) is the conversion of its inactive precursor, big ET-1, into the mature form by the action of specific, phosphoramidon-sensitive, endothelin converting enzyme(s) (ECE). Disparate effects of big ET-1 and ET-1 on renal tubule function suggest that big ET-1 might directly influence renal tubule function. Therefore, the role of the enzymatic conversion of big ET-1 into ET-1 in eliciting the functional response (generation of 1,2-diacylglycerol) to big ET-1 was studied in the rat proximal tubules. 2. In renal cortical slices incubated with big ET-1, pretreatment with phosphoramidon (an ECE inhibitor) reduced tissue immunoreactive ET-1 to a level similar to that of cortical tissue not exposed to big ET-1. This confirms the presence and effectiveness of ECE inhibition by phosphoramidon. 3. In freshly isolated proximal tubule cells, big ET-1 stimulated the generation of 1,2-diacylglycerol (DAG) in a time- and dose-dependent manner. Neither phosphoramidon nor chymostatin, a chymase inhibitor, influenced the generation of DAG evoked by big ET-1. 4. Big ET-1-dependent synthesis of DAG was found in the brush-border membrane. It was unaffected by BQ123, an ETA receptor antagonist, but was blocked by bosentan, an ETA.B-nonselective endothelin receptor antagonist. 5. These results suggest that the proximal tubule is a site for the direct effect of big ET-1 in the rat kidney. The effect of big ET-1 is confined to the brush-border membrane of the proximal tubule, which may be the site of big ET-1 sensitive receptors.

Topics: Animals; Bosentan; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Glycopeptides; Kidney Tubules, Proximal; Male; Microvilli; Protein Precursors; Rats; Rats, Wistar; Receptors, Endothelin; Sulfonamides

Topics: Animals; Cattle; Cell Membrane; Cells, Cultured; Culture Media, Conditioned; Culture Media, Serum-Free; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Kinetics; Polymerase Chain Reaction; Protease Inhibitors; Protein Precursors; Protein Processing, Post-Translational; Pulmonary Artery; Transcription, Genetic; Tumor Necrosis Factor-alpha

The results of early acute haemodynamic studies with anti-endothelin agents are promising. Much still needs to be done, however, before endothelin antagonism is established as a therapeutic strategy in heart failure. We need to know, for example, whether the haemodynamic effects of anti-endothelin drugs are sustained. We need to ensure that there is no reflex activation of other neuroendocrine systems and, preferably, to demonstrate neuroendocrine suppression. Characterisation of the renal actions of endothelin receptor antagonists will also be important. Perhaps the most pressing issue in the development of these agents is elucidation of the role of the endothelial ETB receptor in heart failure. It is now clearly shown that vascular smooth muscle ETB receptors can mediate vasoconstriction in human blood vessels and that these receptors may be particularly important in heart failure. The effect of selective ETB receptor blockade in humans in vivo is not currently known, however, and whether endothelial ETB receptors might tonically offset ETA and ETB receptor mediated smooth muscle contraction remains conjectural. This question is directly relevant to whether selective ETA or non-selective ETA and ETB receptor antagonism might be the better therapeutic strategy in heart failure. ECE inhibition may become another therapeutic option in due course, but at present no specific and selective inhibitors of the enzyme have been developed. The recent demonstration that the selective ETA receptor antagonist BQ-123 improves long term survival in rats with heart failure induced by myocardial infarction suggests that anti-endothelin strategies may hold great therapeutic promise in heart failure.

Topics: Aspartic Acid Endopeptidases; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; Heart Failure; Humans; Metalloendopeptidases; Peptides, Cyclic; Protease Inhibitors

Functional conversion of big-endothelin-1 to endothelin-1 and characterization of endothelin receptor subtype were investigated in cultured rat aortic endothelial cells. Exogenous endothelin-1 and big-endothelin-1 both increased arachidonic acid release and inositol phosphate production dose dependently. Endothelin-1 was more potent than big-endothelin-1 as indicated by EC50 values: 0.5 +/- 0.1 nM and 10.0 +/- 2.0 nM for endothelin-1-induced arachidonic acid release and inositol phosphate formation, respectively, versus 1.0 +/- 0.4 nM and 35.0 +/- 6.0 nM for big-endothelin-1-induced responses. Big-endothelin-1, but not endothelin-1 actions were inhibited by phosphoramidon. Comparative studies of endothelin receptor agonists and antagonists showed that endothelin-3 but not sarafotoxin S6c stimulated arachidonic acid release and inositol phosphate formation. The responses to big-endothelin-1 and endothelin-1 were specifically inhibited by the selective endothelin ETA receptor antagonist, [cyclo-D-Trp-D-Asp-Pro-D-Val-Leu] (BQ-123) but not by the selective endothelin ETB receptor antagonist [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma- methyl-Leu-D-Trp-(COMe)-D-NLeu-ONa] (BQ-788). [125I]Endothelin-1 binding was inhibited by endothelin-1, endothelin-3 and BQ-123 but not by BQ-788. These results indicate that the pharmacological responses to big-endothelin-1 in aortic endothelial cells are due to the extracellular phosphoramidon-sensitive conversion to endothelin-1. Endothelin effects are mediated through endothelin ETA receptors in these cells.

Topics: Animals; Aorta, Thoracic; Arachidonic Acid; Binding, Competitive; Cells, Cultured; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Inositol Phosphates; Protein Precursors; Rats; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin

The formation of endothelins (ET) from exogenous big-ET-1, -2 and -3 was investigated in cultured guinea pig Clara cells. When Clara cells were incubated with 10(-9) or 10(-8) M human big-ET-1 (1-38), the release of ET was 69.9 +/- 7.6 and 221.6 +/- 25 fmol/ml respectively, after 1 h incubation period. Treatment of Clara cells with 1 mM phosphoramidon strongly suppressed (80%) the conversion of big-ET-1 to ET-1 during 1, 2 and 6 h incubation periods. Treatment of Clara cells with thiorphan (1 mM), an inhibitor of neutral endopeptidase, decreased by 44 and 26% the levels of immunoreactive endothelin (ir-ET) in the cell supernatant respectively, after 1 and 2 h incubation periods. When Clara cells were incubated with 10(-8) M human big-ET-2 (1-38), the release of ir-ET was 287.6 +/- 8.9 fmol/ml after a 1 h incubation period. Phosphoramidon and thiorphan (1 mM) reduced the amount of ir-ET generated from exogenous human big-ET-2 (10(-8) M) by 69 and 56%, respectively. When Clara cells were incubated with 10(-8) M human big-ET-3 (1-41), the release of ET was not increased after a 1 h incubation period. Our results suggest that guinea pig Clara cells convert exogenous big-ET-1 and -2 but not big-ET-3 into ET-1 and ET-2, respectively, through metalloproteinases sensitive to both phosphoramidon and thiorphan.

Topics: Animals; Bronchi; Cells, Cultured; Endothelin-1; Endothelins; Epithelial Cells; Epithelium; Glycopeptides; Guinea Pigs; Metalloendopeptidases; Protease Inhibitors; Protein Precursors; Radioimmunoassay; Thiorphan

Treatment of animals with big endothelin-1 (bET) causes pulmonary hypertension and bronchoconstriction, both in vivo and in perfused lungs. The biological activity of bET requires proteolytic cleavage to ET-1 by endothelin converting enzymes (ECE) and possibly other proteases such as neutral endopeptidase 24.11 (NEP 24.11). Since the role of NEP 24.11 in the physiological activation of bET is unclear, we investigated the effects of the selective NEP 24.11 inhibitor thiorphan on bET-induced vaso- and bronchoconstriction in the isolated perfused rat lung. We also studied the effects of phosphoramidon and (S)-2-biphenyl-4-yl-1-(1H-tetraol-5-yl)-ehtylaminomethylphosphonic acid (CGS-26303), i.e. agents which block not only NEP 24.11 but also ECE. The bET-induced vasoconstriction was much less prominent than the bronchoconstriction, i.e. after exposure for 110 min vascular and airway conductance were decreased by 33% and 80% respectively. The small bET-induced vasoconstriction was attenuated to a similar degree by pretreatment with any of the three protease inhibitors. However, thiorphan up to a concentration of 10 microM had only little effect on the bET-induced bronchoconstriction, while 10 microM phosphoramidon or CGS-26303 provided half-maximal and 100 microM phosphoramidon complete protection in this model. This profile of inhibitor action suggests that in rat lung ECE is the major enzyme responsible for activation of bET.

Topics: Airway Resistance; Analysis of Variance; Animals; Aspartic Acid Endopeptidases; Bronchoconstriction; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Female; Glycopeptides; Lung; Metalloendopeptidases; Neprilysin; Organophosphonates; Perfusion; Protease Inhibitors; Protein Precursors; Rats; Rats, Wistar; Tetrazoles; Thiorphan; Vascular Resistance; Vasoconstriction

Endothelin-1 (ET) is a potent endogenous vasoconstrictor which has been shown to be increased following ischaemia and cardiopulmonary bypass. We tested the hypothesis that inhibition of ET synthesis during cardioplegic arrest using phosphoramidon (an ET converting enzyme inhibitor) or blockade of ET receptors using bosentan (a mixed ET(A)/ET(B) antagonist), might improve the postischaemic recovery of coronary flow.. Using an isolated Langendorff perfused rat heart model we compared the addition of phosphoramidon or bosentan to St Thomas' Hospital No. cardioplegia vs. control (plain cardioplegia). We measured recovery of coronary flow following 4 h of cardioplegic arrest at 4 degrees C. In a second series of experiments using an isolated working rat heart model we measured the recovery of cardiac function following 4 h of cardioplegic arrest at 4 degrees C. Results are expressed as percentages of preischaemic values (+/- S.E.M).. In the first series of experiments, addition of phosphoramidon to cardioplegia improved the postischaemic recovery of coronary flow after 30 min of reperfusion: control 81.3% (+/- 3.5); phosphoramidon 10(-6) M 86.2% (+/- 3.1); phosphoramidon 10(-5) M 95.0% (+/- 3.0) P = 0.03 vs. control. Likewise, addition of bosentan 10(-5) M improved coronary flow following 20 min of reperfusion: control 96.7% (+/- 4.0), and bosentan 109.6% (+/- 4.7) P = 0.04. The addition of phosphoramidon or bosentan had no effect on the postischaemic recovery of mechanical function following 30 min of reperfusion.. Both inhibition of ET synthesis and ET receptor blockade during prolonged hypothermic arrest improves postischaemic coronary flow, but appears to have no effect on the recovery of cardiac mechanical function.

Topics: Animals; Bicarbonates; Bosentan; Calcium Chloride; Cardioplegic Solutions; Coronary Circulation; Endothelin Receptor Antagonists; Endothelin-1; Glycopeptides; Heart Arrest, Induced; Hypothermia, Induced; Magnesium; Male; Metalloendopeptidases; Myocardial Contraction; Myocardial Reperfusion Injury; Potassium Chloride; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Sodium Chloride; Sulfonamides; Time Factors

Monocyte deposition on the endothelium is the initial step in atherogenesis. Oxidized low density lipoprotein (Ox-LDL) is involved in the development of the fatty streak which progresses to the atherosclerotic lesion. Our interest focussed on the question, does the endothelium react to Ox-LDL to produce humoral substances that might influence the migration of human blood monocytes? Chemotaxis of monocytes was assessed by the modified membrane-filter technique based on the Boyden chamber principle. Exposure of porcine aorta endothelial cells (ECs) to Ox-LDL (100 micrograms/ml) increased the directional migration of monocytes by 25% (p < 0.01) over that of ECs in the absence of Ox-LDL. Radioimmunoassay of the EC culture media revealed the presence of immunoreactive endothelin-1 (ir-ET-1). The endothelin converting enzyme inhibitor, phosphoramidone (10 microM), when incubated together with ECs and Ox-LDL, suppressed the synthesis of ir-ET-1 by 53% (p < 0.05) and the migration decreased by 12% (p < 0.05). Preincubation of monocytes with the ETA receptor-selective antagonist, BQ-123 (1 microM), followed by exposure to ECs plus Ox-LDL, lead to a decrease in their migration by 12% (p < 0.05) compared to monocytes not treated with BQ-123. These results show that Ox-LDL acts on ECs to enhance the synthesis of ir-ET-1 which in turn increases the directional migration of monocytes. Phosphoramidone decreased the synthesis of ir-ET-1 but migration was affected only modestly; monocyte ETA receptor blockade by BQ-123 also suppressed migration toward EC chemoattractants to a small extent. Both results suggest that in addition to ir-ET-1 other chemotactic factors are being released by the ECs; Ox-LDL appears to enhance their release or synthesis.

Topics: Animals; Aorta; Aspartic Acid Endopeptidases; Cells, Cultured; Chemotaxis, Leukocyte; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Glycopeptides; Humans; Lipoproteins, LDL; Metalloendopeptidases; Monocytes; Oxidation-Reduction; Peptides, Cyclic; Protease Inhibitors; Radioimmunoassay; Swine

Because nitric oxide (NO.) and endothelin (ET)-1 frequently have opposing effects on physiological and inflammatory processes, we sought to determine whether ET-1 regulates NO. synthesis by the inducible isoform of NO. synthase (iNOS). L2 cells are a rat lung epithelial cell line that synthesizes ET-1 and in which ET-1 has an autocrine role. In the current study, we demonstrate that L2 cells generate the oxidative products of NO., nitrite and nitrate, after exposure to tumor necrosis factor-alpha, lipopolysaccharide, and interferon-gamma. Exposure to these cytokines also dramatically increases the expression of iNOS mRNA. NG-monomethyl-L-arginine, dexamethasone, and cycloheximide prevent the cytokine-mediated increase in NO. oxidative products, demonstrating that iNOS accounts for their generation. Because L2 cells synthesize ET-1, to test the effect of removing endogenous ET-1, we used phosphoramidon (an ET-converting enzyme inhibitor) or BQ-123 (an ET receptor A antagonist). Removal of endogenous ET-1 with either phosphoramidon or BQ-123 significantly augments cytokine-stimulated NO. synthesis by approximately 20%. To further test the effect of ET-1 on iNOS, we treated cells with phosphoramidon to inhibit endogenous ET-1 synthesis and then administered ET-1 (10(-9) to 10(-7) M). In this setting, ET-1 significantly decreases inducible NO. production by 33% and iNOS mRNA by 50%. We conclude that ET-1 can decrease inducible NO. synthesis by cytokine-stimulated lung epithelial cells.

Topics: Animals; Cell Line; Cycloheximide; Cytokines; Dexamethasone; DNA Primers; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Induction; Enzyme Inhibitors; Epithelium; Glycopeptides; Humans; Lung; Nitrates; Nitric Oxide Synthase; Nitrites; omega-N-Methylarginine; Peptides, Cyclic; Polymerase Chain Reaction; Rats; Receptor, Endothelin A; Recombinant Proteins; RNA, Messenger; Transcription, Genetic

Experiments on isolated strips of rat v. portae revealed that endothelin participates in the development of myogenic reactions of vascular smooth muscles. Endothelin is known to be released by endotheliocytes and to stimulate the contraction of vascular smooth muscle cells. Rats with inherited arterial hypertension showed significant decrease of the stimulating effect of endothelin-1 upon the contraction of vascular smooth muscles. At the same time, the effect of phosphoramidon, an inhibitor of endothelin-converting enzyme, upon the length-tension curve of the strips of v. portae is more marked in hypertensive vs. control rats. The data obtained testify to the fact that the increase of vascular tone in the hypertensive rats is most likely to be conditioned by the involvement of endothelin.

Topics: Acetylcholine; Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hypertension; In Vitro Techniques; Metalloendopeptidases; Muscle Contraction; Muscle, Smooth, Vascular; Portal Vein; Protease Inhibitors; Rats; Rats, Inbred SHR

1. We have studied the conversion of big endothelin-1 (big ET-1), big endothelin-2 (big ET-2) and big endothelin-3 (big ET-3) and characterized the enzyme involved in the conversion of the three peptides in guinea-pig lung parenchyma (GPLP). 2. Endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) (10 nM to 100 nM) caused similar concentration-dependent contractions of strips of GPLP. 3. Big ET-1 and big ET-2 also elicited concentration-dependent contractions of GPLP strips. In contrast, big ET-3, up to a concentration of 100 nM, failed to induce a contraction of the GPLP. 4. Incubation of strips of GPLP with the dual endothelin converting enzyme (ECE) and neutral endopeptidase (NEP) inhibitor, phosphoramidon (10 microM), as well as two other NEP inhibitors thiorphan (10 microM) or SQ 28,603 (10 microM) decreased by 43% (P < 0.05), 42% (P < 0.05) and 40% (P < 0.05) the contractions induced by 30 nM of big ET-1 respectively. Captopril (10 microM), an angiotensin-converting enzyme inhibitor, had no effect on the contractions induced by big ET-1. 5. The incubation of strips of GPLP with phosphoramidon (10 microM), thiorphan (10 microM) or SQ 28,603 (10 microM) also decreased by 74% (P < 0.05), 34% and 50% (P < 0.05) the contractions induced by 30 nM big ET-2 respectively. As for the contractions induced by big ET-1, captopril (10 microM) had no effect on the concentration-dependent contractions induced by big ET-2. 6. Phosphoramidon (10 microM), thiorphan (10 microM) and SQ 28,603 (10 microM) significantly potentiated the contractions of strips of GPLP induced by both ET-1 (30 nM) and ET-3 (30 nM). However, the enzymatic inhibitors did not significantly affect the contractions induced by ET-2 (30 nM) in this tissue. 7. These results suggest that the effects of big ET-1 and big ET-2 result from the conversion to ET-1 and ET-2 by at least one enzyme sensitive to phosphoramidon, thiorphan and SQ 28,603. This enzyme corresponds possibly to EC 3.4.24.11 (NEP 24.11) and could also be responsible for the degradation of ETs in the GPLP.

Topics: Alanine; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Endothelin-1; Endothelins; Glycopeptides; Guinea Pigs; Histamine; In Vitro Techniques; Lung; Neprilysin; Protease Inhibitors; Protein Precursors; Thiorphan

Endothelin converting enzyme (ECE) from intact cells of a permanent human endothelial cell line, EA.hy926, was studied by examining the effects of phosphoramidon, an endothelin converting enzyme inhibitor, on the levels of secreted endothelin-1 and big endothelin-1. The specific ECE activity was demonstrated by a phosphoramidon dose-dependent decrease in ET-1 level with a concomitant increase in big ET-1 level. By using a specific neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, it was also shown that the phosphoramidon-sensitive ET-1 degrading activity in this cell line is due to the NEP 24.11 activity. Other serine, acid, and cysteine protease inhibitors had no effect on the endogenous synthesis of ET-1 and big ET-1 supporting the evidence that ECE is insensitive to these protease inhibitors as has been demonstrated with the isolated enzyme.

Topics: Aspartic Acid Endopeptidases; Cells, Cultured; Endometrium; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; Female; Glycopeptides; Humans; Metalloendopeptidases; Protein Precursors; Radioimmunoassay; Thiorphan

To investigate the role of endogenous endothelin-1 (ET-1) expression and its interaction with the ETA receptor in the physiologic regulation of vascular tone as well as in the development of abnormal retinal hemodynamics in diabetes.. Retinal blood flow, using digitized video fluorescein angiography recordings, was quantitated after intravitreous injections of ET-1; BQ-123, an ETA receptor antagonist; and phosporamindon, an endothelin converting enzyme inhibitor in the eyes of diabetic and nondiabetic rats. A total of 154 rats were used for these experiments. Message levels of preproendothelin-1 (preproET-1) were measured from the retina of diabetic and nondiabetic rats using competitive polymerase chain reaction (PCR) techniques.. Retinal blood flow was reduced (33%, P < 0.001) in diabetic rats compared to nondiabetic rats. BQ-123, an ETA receptor antagonist, but not saralasin, an angiotensin receptor antagonist, increased retinal blood flow in a dose-dependent manner in diabetic (EC50 of 8 x 10(-7) M) and in nondiabetic rats (EC50 of 8 x 10(-8) M). Besides being resistant to BQ-123, the maximal response in diabetic animals occurred 20 minutes later than in nondiabetic animals. Decreasing ET-1 levels by inhibiting endothelin-converting enzyme with phosphoramidon normalized retinal blood flow in diabetic rats. In nondiabetic rats, the intravitreous injection of exogenous ET-1 (10(-8) M) resulted in retinal blood flow decreases comparable to those measured in diabetic animals, and the subsequent injection of 10(-4) M BQ-123 produced retinal blood flow changes comparable to those measured in BQ-123 injected diabetic rats. Comparison of preproET-1 messenger RNA expression in the retina, brain and lung of control and diabetic rats using quantitative PCR and Northern blot analysis showed 2.0- and 1.7-fold increases in the retina and the brain, respectively, without changes in the lung.. These data suggest that ET-1 is involved in the regulation of retinal blood flow in normal physiologic outcome, and an increase in the endogenous expression of ET-1 contributes to the reduction of retinal blood flow reported in the early stages of diabetes mellitus.

Topics: Animals; Brain; Diabetes Mellitus, Experimental; Diabetic Retinopathy; DNA Primers; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Fluorescein Angiography; Glycopeptides; Hemodynamics; Male; Metalloendopeptidases; Peptides, Cyclic; Polymerase Chain Reaction; Protease Inhibitors; Protein Precursors; Rats; Rats, Inbred BN; Rats, Sprague-Dawley; Receptors, Endothelin; Retina; Retinal Vessels; RNA, Messenger

The effect of big endothelin-1 (big ET-1) and its conversion to endothelin-1 (ET-1) in rabbit cerebral arteries were examined. Big ET-1 and ET-1 induced concentration-dependent contractions in the basilar artery; ET-1 was approximately 8 times more potent than big ET-1. The metalloprotease inhibitor phosphoramidon (30 mumol/l) almost abolished the contractile response to big ET-1, whereas the ET-1-induced contraction was unaffected. Removal of the endothelium did not attenuate the big ET-1-induced contraction. ET-1 was approximately 14 times more potent than endothelin-3 (ET-3) to elicit contraction. The contractions induced by big ET-1, ET-1 and ET-3 were all inhibited by ET(A) receptor antagonist BQ 123 (3 mumol/l). The ET(B) receptor antagonist IRL 1038 (3 mumol/l) had no effect on the contractile responses to big ET-1 and ET-1, but produced a small inhibition of the ET-3-induced contraction. Formation of ET-1 was demonstrated in membrane fractions of cerebral arteries incubated with big ET-1 as measured by high pressure liquid chromatography followed by radioimmunoassay. These results suggest that externally applied big ET-1 is converted to ET-1 by a phosphoramidon-sensitive "endothelin converting enzyme" present in the vascular smooth muscle cells. The ET-1 formed subsequently mediates the big ET-1-induced contraction by activation of mainly ET(A) receptors, although a small contribution of ET(B) receptors cannot be excluded.

Topics: Animals; Cerebral Arteries; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-3; Endothelins; Female; Glycopeptides; Male; Metalloendopeptidases; Muscle Contraction; Muscle, Smooth, Vascular; Peptide Fragments; Peptides, Cyclic; Protein Precursors; Rabbits; Receptors, Endothelin

The intratracheal (i.t.) instillation of Sephadex beads into rat induced inflammation and a 30-fold increase in the endothelin-1-like immunoreactivity (ET-1-LI) of broncho-alveolar lavage fluid. The levels were highest 24 h after the instillation and had declined significantly after 48 h. At a dose of 1 mg kg-1 i.t., the glucocorticosteroid budesonide almost abolished this response. Phosphoramidon, which inhibits neutral endopeptidase, an enzyme reported to degrade ET-1 and also to inhibit the endothelin-converting enzyme, potentiated the Sephadex-induced rise in ET-1-LI. Chymostatin and heparin, which are reported to reduce the formation of ET-1, did not affect the increase in ET-1-LI. The present model represents a very reactive system for analyzing the changes in ET-1 levels during inflammation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Budesonide; Chromatography, High Pressure Liquid; Dextrans; Endothelin-1; Enzyme Inhibitors; Glycopeptides; Heparin; Hydrogen Peroxide; Male; Oligopeptides; Pneumonia; Pregnenediones; Rats; Rats, Sprague-Dawley

Alpha-naphthylthiourea when injected intraperitoneally to rats (10 mg kg-1 i.p.) produced lung oedema as indicated by an increase in lung weight/body ratio and pleural effusion reaching a maximum within 4 hours. Prior intravenous single bolus injection of endothelin-1 elicited a significant and dose-dependent inhibition in both parameters. However, prior i.v. injection of angiotensin II using relatively higher doses did not alter the oedema-producing effect of alpha-naphthylthiourea indicating a characteristic for endothelin-1. The inhibitory effect of endothelin-1 on pleural effusion is more prominent than lung weight/body weight ratio. The resolution of lung oedema by single bolus i.v. injection of endothelin-1 is probably due to the acute long-lasting and potent vasoconstrictor effect of the peptide and its large accumulation in lung tissue. Phosphoramidon, an inhibitor of endothelin converting enzyme, did not alter the oedema producing effect of alpha-naphthylthiourea indicating the lack of the participation of endothelin-peptide cascade to this pathological event. Bosentan, a non-selective receptor blocker of endothelin-1, did not inhibit the preventive effect of the peptide against alpha-naphthylthiourea-induced lung oedema. Possible mechanisms of the acute effect of endothelin-1 on lung oedema are discussed.

Topics: Angiotensin II; Animals; Aspartic Acid Endopeptidases; Body Weight; Bosentan; Endothelin-1; Endothelin-Converting Enzymes; Glycopeptides; Male; Metalloendopeptidases; Organ Size; Pleural Effusion; Protease Inhibitors; Pulmonary Edema; Rats; Sulfonamides; Thiourea

Diaspirin cross-linked hemoglobin (DCLHb) is a promising hemoglobin-based, oxygen-carrying resuscitative solution. DCLHb (400 mg/kg, iv) produces significant cardiovascular effects, along with an increase in plasma endothelin-1 (ET-1) level, when administered to conscious or anesthetized rats. Present studies were performed to determine whether the cardiovascular effects of DCLHb are due to an increase in the conversion of proendothelin-1 (1-38) (proET-1) to ET-1 by endothelin-converting enzyme (ECE). The regional circulatory and systemic hemodynamic effects of proET-1 (20 micrograms/kg, iv) and DCLHb (400 mg/kg, iv) were determined by using a radioactive microsphere technique in control rats and rats pretreated with phosphoramidon (ECE inhibitor). Administration of proET-1 produced an immediate increase in mean arterial pressure (MAP)(52%) and total peripheral resistance (TPR) (55%); stroke volume (SV) and cardiac output were not affected in the initial phase but were decreased subsequently. Heart rate (HR) was not affected after administration of proET-1. A significant increase in blood flow to the heart (39%), brain (46%), kidneys (74%), portal system (40%), and gastrointestinal tract (GIT) (42%) was also observed after administration of proET-1. Vascular resistance was found to be significantly increased in the mesentery and pancreas (168%) and in the musculoskeletal system (147%) and decreased in the kidneys (-11%) after administration of proET-1. Phosphoramidon (4 mg/kg, iv) pretreatment attenuated the increase in MAP and TPR induced by proET-1. Phosphoramidon pretreatment significantly attenuated the proET-1-induced increase in blood flow to the heart, brain, kidneys, portal system, and GIT. The increase in vascular resistance induced by proET-1 in the mesentery and pancreas and in the musculoskeletal system was also attenuated by phosphoramidon. DCLHb increased MAP (63%) and TPR (54%) without affecting HR. DCLHb increased blood flow to the heart (95%), GIT (45%), portal system (43%), and skin (79%) and increased vascular resistance in the musculoskeletal system (58%). In phosphoramidon-treated rats, DCLHb increased MAP (99%), HR (25%), cardiac output (37%), and TPR (60%). DCLHb increased blood flow to the heart (104%), brain (66%), kidneys (49%), GIT (59%), portal system (63%), and skin (100%) when administered to phosphoramidon-treated rats. Phosphoramidon did not attenuate any of the DCLHb-induced cardiovascular effects. It is concluded that pro

Topics: Animals; Aspartic Acid Endopeptidases; Aspirin; Blood Gas Analysis; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hemodynamics; Hemoglobins; Male; Metalloendopeptidases; Protease Inhibitors; Protein Precursors; Rats; Rats, Sprague-Dawley; Regional Blood Flow

Using cultured human aortic endothelial cells, we examined the effects of phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, on the release of endogenous endothelin-1 (ET-1) and big endothelin-1 (big ET-1), and on the generation of ET-1 from exogenously applied big ET-1. Phosphoramidon, at concentrations of 10(-6) to 2 x 10(-4) M, caused a biphasic alteration of the ET-1 release, i.e., at lower concentrations of the drug, there were slight but unexpected increases of the release, whereas higher concentrations led to a decrease which is due to the drug-induced inhibition of ECE. The former effect appears to be based on the inhibition of ET-1 degradation by neutral endopeptidase 24.11 (NEP), since kelatorphan, a specific NEP inhibitor, produced a similar increasing effect on ET-1 release. Phosphoramidon enhanced the big ET-1 release from the cells in a concentration-dependent manner. When high concentrations of phosphoramidon were added, there was a dramatic increase in the release of big ET-1, which cannot be explained only by the drug-induced inhibition of ECE. This increase in big ET-1 release appeared to be partly due to a transient stimulation of the expression of prepro ET-1 mRNA. The amount of ET-1 generated from exogenously applied big ET-1 was markedly decreased by phosphoramidon in a concentration-dependent manner. In a similar fashion, phosphoramidon markedly inhibited ECE activity of the membrane fraction of cultured cells. Thus, ET-1 generation from exogenously applied big ET-1 reflects the functional phosphoramidon-sensitive ECE activities in human aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Aorta; Aspartic Acid Endopeptidases; Cell Membrane; Cells, Cultured; Dipeptides; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Metalloendopeptidases; Protein Precursors; RNA, Messenger; Sensitivity and Specificity; Stimulation, Chemical

The effects of diltiazem and phosphoramidon on sudden death induced by endothelin (ET)-1 and by big ET-1 were compared in rodents. Diltiazem (2 mg/kg, i.v.) remarkably diminished the lethal toxicity of ET-1 with a reduction in the extent of the rise in plasma immunoreactive ET-1-like activity (IR-ET-1), tissue IR-ET-1 accumulation in the heart and the rise in plasma potassium concentration. In big ET-1-induced lethality, diltiazem only slightly prolonged the latency and did not reduce the mortality. Although diltiazem moderately inhibited the rise in plasma IR-ET-1 and potassium concentration in these mice, it did not affect the accumulation of IR-ET-1 in the heart, lung or kidney. Phosphoramidon (2 mg/kg, i.v.) decreased the lethality of big ET-1 with the decrement in elevation of IR-ET-1 in the heart, lung and plasma as well as with the decrease in plasma potassium concentration, but it failed to improve any parameters in ET-1-induced lethality. In anesthetized rats, ET-1 (5 nmol/kg, i.v.) elevated ST-segment of electromyocardiograms, and diltiazem (2 mg/kg, i.v.) significantly reversed this change. Big ET-1 (25 nmol/kg, i.v.) also induced the ST-segment elevation, which was significantly inhibited by phosphoramidon but not by diltiazem. These findings suggest that accumulation of ET-1 in the heart, which may lead to lethal cardiac ischemia, is an important factor in the lethality of ET-1, while additional factors (such as hemoconcentration and bronchoconstriction) may be involved in big ET-1-induced lethality.

Topics: Anesthesia; Animals; Death, Sudden, Cardiac; Diltiazem; Electrocardiography; Endothelin-1; Endothelins; Glycopeptides; Kidney; Lung; Male; Mice; Mice, Inbred ICR; Myocardial Ischemia; Myocardium; Potassium; Protease Inhibitors; Protein Precursors

1. In the present study, the precursors of endothelin-1, endothelin-2 and endothelin-3 were tested for their pressor and bronchoconstrictor properties in the anaesthetized guinea-pig. In addition, the effects of big-endothelin-1 and endothelin-1 were assessed under urethane or ketamine/xylazine anaesthesia. 2. When compared to ketamine/xylazine, urethane markedly depressed the pressor and bronchoconstrictor properties of endothelin-1 and big-endothelin-1. 3. Under ketamine/xylazine anaesthesia, the three endothelins induced a biphasic increase of mean arterial blood pressure. In contrast, big-endothelin-1, as well as big-endothelin-2 (1-38), induced only sustained increase in blood pressure whereas big-endothelin-3 was inactive at doses up to 25 nmol kg-1. 4. Big-endothelin-1, but not big-endothelin-2, induced a significant increase in airway resistance. Yet, endothelin-1, endothelin-2 and endothelin-3 were equipotent as bronchoconstrictor agents. 5. Big-endothelin-1, endothelin-1 and endothelin-2, but not big-endothelin-2, triggered a marked release of prostacyclin and thromboxane A2 from the guinea-pig perfused lung. 6. Our results suggest the presence of a phosphoramidon-sensitive endothelin-converting enzyme (ECE) which is responsible for the conversion of big-endothelin-1 and big-endothelin-2 to their active moieties, endothelin-1 and 2. However, the lack of bronchoconstrictor and eicosanoid-releasing properties of big-endothelin-2, as opposed to endothelin-2 or big-endothelin-1, suggests the presence of two distinct phosphoramidon-sensitive ECEs in the guinea-pig. The ECE responsible for the systemic conversion of big-endothelins possesses the same affinity for big-endothelin-l and 2 but not big-endothelin-3. In contrast, in the pulmonary vasculature is localized in the vicinity of the sites responsible for eicosanoid release, an ECE which converts more readily big-endothelin-1 than big-endothelin-2.

Topics: Analysis of Variance; Animals; Aspartic Acid Endopeptidases; Blood Pressure; Bronchoconstriction; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Epoprostenol; Female; Glycopeptides; Guinea Pigs; Humans; In Vitro Techniques; Ketamine; Lung; Male; Metalloendopeptidases; Muscle, Smooth; Protease Inhibitors; Protein Precursors; Thromboxane A2; Urethane; Xylazine

Both endothelins and their big-endothelin precursors were found capable of inducing the release of arachidonic acid from purified cultures of rat astrocytes. Their order of potency was as follows: big-endothelin-3 < big-endothelin-1 < endothelin-1 = endothelin-3. Mature endothelins induced the release of arachidonic acid in a rapid fashion. In contrast, much longer incubation times were required for big-endothelins to exert an effect, suggesting that their activity was dependent on their conversion. When big-endothelin-1 was added to the incubation medium of intact live astrocytes, it was converted into mature endothelin-1 in a time-dependent manner and the conversion was inhibited by phosphoramidon. This suggests that astrocytic endothelin-converting enzyme is (at least in part) an external membrane-bound metalloprotease. Some conversion of big-endothelin-3 into endothelin-3 also occurred. However, it was less efficient than the conversion of big-endothelin-1, which is compatible with the lower bioactivity of big-endothelin-3 vs. that of big-endothelin-1 in astrocytes.

Topics: Animals; Arachidonic Acid; Aspartic Acid Endopeptidases; Astrocytes; Binding, Competitive; Cells, Cultured; Diencephalon; Dose-Response Relationship, Drug; Drug Interactions; Endothelin-1; Endothelin-3; Endothelin-Converting Enzymes; Endothelins; Frontal Lobe; Glycopeptides; Metalloendopeptidases; Protease Inhibitors; Protein Precursors; Radioligand Assay; Rats

Subcellular fractionation of the phosphoramidon sensitive membrane-bound endothelin converting enzyme (ECE-1) activity from homogenates of bovine aortic endothelial cells and the human endothelial cell line EA.hy 926, combined with studies of intact cells, shows ECE-1 to be localised primarily to the plasma membrane with the topology of an ectoenzyme. To overcome the problem of neutral endopeptidase 24.11 contaminating the human ECE-1 activity solubilised from the plasma membrane fractions of EA.hy 926, we have used isoelectric focusing to simultaneously solubilise and separate these activities. The metallopeptidase ECE-1 obtained displayed a neutral pH optimum, a molecular weight of 250 kDa on gel filtration chromatography and was inhibited by phosphoramidon with an IC50 of 0.8 microM.

Topics: Animals; Aorta; Aspartic Acid Endopeptidases; Cattle; Cell Fractionation; Cell Line; Cells, Cultured; Centrifugation, Density Gradient; Chromatography, Gel; Chromatography, Ion Exchange; Cross Reactions; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Hydrogen-Ion Concentration; Kinetics; Metalloendopeptidases; Neprilysin; Peptidyl-Dipeptidase A; Protein Precursors; Radioimmunoassay; Subcellular Fractions; Umbilical Veins

The enzyme responsible for the conversion of exogenous big endothelin-1 to endothelin-1 by porcine coronary arterial smooth muscle has been shown to be a metalloprotease. The potencies of eight metalloprotease inhibitors for this endothelin-converting enzyme were determined. CGS 25015, CGS 26129, and thiorphan inhibited the enzyme activity monophasically with IC50 values of 2.6, 2.4, and 190 microM, respectively. In contrast, the data obtained using phosphoramidon as an inhibitor were best fit by a two-site model. The biphasic concentration-response curve had IC50 values of 4.6 microM and 2.2 mM. Three analogs of phosphoramidon were also tested for enzyme inhibition. Removal of the rhamnose moiety of phosphoramidon reduced the potency (IC50 = 15 microM), whereas substitution of the rhamnose by N-[2-(2-naphthyl)ethyl] improved the potency (IC50 = 2.0 microM). These results identify a thiol and a phosphonyl series of compounds as smooth muscle endothelin-converting enzyme inhibitors. The structure-activity relationships revealed that an aromatic or aliphatic group in the P2' position or an aromatic group in the P1 position of the inhibitor significantly increased the potency.

Topics: Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Activation; Glycopeptides; Metalloendopeptidases; Methionine; Muscle, Smooth, Vascular; Propionates; Protein Precursors; Structure-Activity Relationship; Swine; Thiorphan

Compared with endothelin-1 (ET-1), big endothelin-1 (big ET-1) is only weakly active on isolated vascular smooth muscle preparations. However, on systemic administration high doses of big ET-1 (1 nmol.kg-1) are approximately equipotent to ET-1, indicating the existence of an endothelin converting enzyme in the circulation that rapidly converts big ET-1 to ET-1. In this study arterial blood levels of big ET-1 and ET-1 immunoreactivity were measured after bolus i.v. administration of big ET-1 (1 or 3 nmol.kg-1) or ET-1 (1 nmol.kg-1) in anaesthetised male Wistar rats. In addition, the effect of phosphoramidon (10 mg.kg-1) on the pressor response to big ET-1 and its disappearance rate from the circulation were examined. After big ET-1 injection, circulating ET-1 concentrations did not exceed 2% of the big ET-1 level. Phosphoramidon reduced the pressor response to big ET-1 by 93%, but did not alter its rate of clearance from the circulation. Thus exogenous big ET-1 is converted locally in the vasculature and its disappearance from the circulation is not dependent on conversion to ET-1.

Topics: Animals; Blood Pressure; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Glycopeptides; Male; Protein Precursors; Radioimmunoassay; Rats; Rats, Wistar

Human and porcine big ET-1 and big ET-2 are similarly potent in contracting parenchymal strips of the guinea-pig lung while big ET-3 is inactive, suggesting that the endothelin-converting enzyme (ECE) which converts big ET-3 is not present and that at least two distinct ECE activities exist, one selective for big ET-1 and big ET-2 and one for big ET-3. Metalloendoprotease inhibitors (phosphoramidon and DL-thiorphan), but not captopril, inhibited the contractions elicited by human big ET-1 and big ET-2 but DL-thiorphan was less active, suggesting that a non-selective enzymatic process is involved in conversion of big ET-1 and big ET-2 in addition to a phosphoramidon-sensitive ECE. Big ET-1 and big ET-2 induced much higher contractions than their corresponding mature peptides. Both metalloendoprotease inhibitors, but not captopril, similarly potentiated contractions induced by ET-1, ET-2 or ET-3 to the level of those evoked by big ET-1 and big ET-2, indicating that only mature ET isopeptides and not their precursors are susceptible to degradation by metalloendoproteases.

Topics: Animals; Captopril; Endopeptidases; Endothelin-1; Endothelins; Glycopeptides; Guinea Pigs; Humans; In Vitro Techniques; Lung; Male; Muscle Contraction; Muscle, Smooth; Protease Inhibitors; Protein Precursors; Structure-Activity Relationship; Swine; Thiorphan

Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.

Topics: Aspartic Acid Endopeptidases; Breast; Breast Neoplasms; Chromatography, High Pressure Liquid; Culture Media; Edetic Acid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Fibroblasts; Glycopeptides; Growth Substances; Humans; Hydrogen-Ion Concentration; Iron Chelating Agents; Kinetics; Metalloendopeptidases; Mitogens; Phenanthrolines; Protease Inhibitors; Protein Precursors; Sensitivity and Specificity; Tumor Cells, Cultured

1. It is generally accepted that endothelial cells secrete endothelin (ET) to the underlying media which mediates the contractile effects of ET. However, there is some evidence that animal vascular smooth muscle cells (VSMCs) also secrete ET. We cultured VSMCs from human vessels representative of a number of different vascular beds to determine whether human VSMCs endogenously secrete ET. 2. VSMCs explanted from adult arterial vessels secrete picomolar quantities of immunoreactive mature ET: coronary artery 226.6 +/- 58.8 pM/10(6) cells (n = 7), thoracic aorta 169.5 +/- 105.4 pM/10(6) cells (n = 3), left internal mammary artery 102.4 +/- 23.1 pM/10(6) cells (n = 3) and saphenous vein 69.4 +/- 19.9 pM/10(6) cells (n = 3), as well as from umbilical vein (HUVSMCs) 38.3 +/- 4.3 pM/10(6) cells (n = 3). Secretion of immunoreactive big ET-1 was also detected: coronary artery 249.1 +/- 59.4 pM/10(6) cells (n = 7), thoracic aorta 120.0 +/- 13.4 pM/10(6) cells (n = 3), left internal mammary artery 170.0 +/- 68.2 pM/10(6) cells (n = 3), saphenous vein 105.1 +/- 30.7 pM/10(6) cells (n = 3) and from umbilical vein 146.3 +/- 7.4 pM/10(6) cells (n = 3). Comparable, intracellular levels of immunoreactive big ET-1 and mature ET were also detected in cultured VSMCs. 3. Since enzyme-dispersed VSMCs are thought to be more differentiated and more closely resemble their in vivo counterparts, and these enzyme-dispersed VSMCs from human umbilical vein (HUVSMCs) also secreted the greatest levels of immunoreactive peptides, they were characterized further. Reverse transcription-polymerase chain reaction assay demonstrated that HUVSMCs express ET-1 mRNA. High performance liquid chromatography coupled to radioimmunoassay revealed that HUVSMCs secrete ET-1 and ET-3, in addition to big ET-1. However, levels of ET are not altered by 100 AM phosphoramidon,an inhibitor of metalloproteases or by 100 microM pepstatin A, an aspartyl protease inhibitor.4. In concordance, KD and Bmax values for [125I]-ET-l saturation binding are not altered in HUVSMC cultures incubated for 24 h with 100 microM phosphoramidon (431 +/- 218 PM and 31.1 +/- 12.7 fmol mg-1;mean =/- s.e.mean, n = 3) or 100 microM pepstatin A (381 +/- 169PM and 19.9 +/- 7.8 fmol mg-1, n = 3) as compared to controls (355 +/- 99 pM and 33.3 +/- 9.3 fmol mg-1; n = 3). This observation indicates the absence of an autocrine 'unmasking' effect for ET receptors.5. HUVSMCs synthesize and secrete immunoreactive ET-1, ET-3 and big ET-1, and p

Topics: Antimalarials; Base Sequence; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Female; Glycopeptides; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Muscle, Smooth, Vascular; Pepstatins; Protease Inhibitors; Protein Precursors; Proteins; Radioimmunoassay; RNA, Messenger; Umbilical Veins

We developed a procedure for individual measurement of big endothelin-1 (big ET-1) and the two products of big ET-1 conversion (ET-1 and C-terminal fragment (CTF) of big ET-1 (big ET-1(22-38)), using selective solid-phase extraction and specific radioimmunoassays. These techniques were used to measure the levels of these peptides in extracts of human plasma after brachial artery infusions of big ET-1. Infusion of big ET-1 (50 pmol/min, n = 6) caused a decrease in forearm blood flow from 3.03 to 1.55 ml/dl/min after 60 min (p < 0.05). In agreement, the levels of plasma immunoreactive (IR) big ET-1, ET, and CTF were significantly increased from basal levels in the infused arm (from undetectable to 386 pM, 2.2-7.5 pM, and 0.17-37 pM, respectively; p < 0.05). In the presence of the metalloprotease inhibitor phosphoramidon (30 nmol/min), the increase in IR-ET and the associated vasoconstriction were abolished. However, IR-CTF was still detected, suggesting that either some conversion by phosphoramidon-insensitive endothelin-converting enzyme (ECE) was occurring and/or that CTF was being preserved from further proteolysis by phosphoramidon. These data confirm that exogenous big ET-1 is locally converted to ET-1 and CTF in the human forearm, at least in part by a phosphoramidon-sensitive ECE. Furthermore, because measurable levels of newly synthesized ET-1 are probably rapidly reduced as a result of receptor binding, the assay of IR-CTF may be a more sensitive measure of the overexpression of ET-1 in disease.

Topics: Adult; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Humans; Male; Metalloendopeptidases; Peptide Fragments; Protein Precursors; Vasoconstriction

1. Phosphoramidon, a potent inhibitor of endopeptidase-24.11 (E-24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin-1 (big ET-1) in rats. To examine whether E-24.11 or thermolysin convert big ET-1 to endothelin-1 (ET-1) and C-terminal fragment (CTF), the effects on porcine and human big ET-1 of each of the purified enzymes were compared in vitro. 2. For E-24.11, the relative rates of hydrolysis were ET-1 > CTF >> big ET-1. The relative half-lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET-1 > 24 h; ET-1, 37 min; CTF, 57 min. For comparison, the half-life for hydrolysis of substance P under similar conditions was 2.1 min. 3. For thermolysin the relative rates of hydrolysis were found to be big ET-1 > CTF > ET-1. The relative half-lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET-1, 25 min; ET-1, 56 min; CTF, 47 min. 4. Because the low rate of conversion of big ET-1 to ET-1 by E-24.11 did not yield sufficient ET-1 for h.p.l.c. quantification a RIA specific for ET-1(16-21) was used to study further the hydrolysis of big ET-1 by E-24.11. Incubation of big ET-1 (0.2-2 nmol) with E-24.11 (4-400 ng) generated ET-1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET-1 (2 nmol) with E-24.11 (40 ng) for 8 h showed that steady state levels of ET-1 were achieved after 4 h indicating that the rate of ET-1 degradation was then equal to the formation of new ET-1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET-1 had been produced, but the yield was insufficient for verification by mass spectrometry.5. Both ET-l-like and CTF-like peaks were detected at 214 nm when the products of big ET-1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET-1 with amounts in the range 120-160 pmol.6. The hydrolysis profile of ET-1 by E-24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue.7. Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E-24.11 and could be useful for the design of ECE inhibitors. Since E-24.11 can both synthesize and hydrolyse ET-1, the presence of E-24.11 in membrane fractions or in partially purified ECE preparations may produce misleading esti

Topics: Animals; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Glycopeptides; Humans; Hydrolysis; Kinetics; Mass Spectrometry; Neprilysin; Peptide Fragments; Protein Precursors; Radioimmunoassay; Substance P; Swine; Thermolysin

1. Endothelin receptors, that mediate contraction of the human isolated coronary artery, were characterized by use of a number of agonists and antagonists. Contraction induced by the non-selective agonists, endothelin (ET)-1 and sarafotoxin S6b, was compared in endothelium-intact and endothelium-denuded ring segments. The effects of ET-1 and BQ-123 (an ETA receptor antagonist) were investigated both in ring segments and in spirally cut strips. Lastly, the effect of phosphoramidon was studied on contraction induced by big-ET-1. 2. The order of agonist potency (pD2) in endothelium-intact coronary artery ring segments was: ET-1 (8.27) approximately sarafotoxin S6b (8.16) > big-ET-1 (< 7.1) approximately ET-3 (< 6.9). [Ala1,3,11,15]ET-1 (ETB receptor agonist) caused significant contraction only at 1 microM, whereas 0.3 microM big-ET-3 had no effect. Removal of the endothelium in ring segments did not affect the contractile response to ET-1 or to sarafotoxin S6b. 3. After a full concentration-response curve had been obtained to ET-1 or sarafotoxin S6b, further contractions of the endothelium-intact coronary artery segments could only be achieved by applying ET-1 in segments exposed to sarafotoxin S6b, and not the reverse. 4. BQ-123 (0.1 microM) antagonized contractions of endothelium-intact ring segments induced by sarafotoxin S6b (pKB 7.86). Only 10 microM BQ-123 antagonized contractions induced by ET-1 (pKB 5.75). FR139317 was also more potent against sarafotoxin S6b (pKB 8.24-8.47) than against ET-1 (pKB 6.11). [Ala1,3,11,15]ET-1 (1 microM) had no effect on the contractile response to ET-1 or to sarafotoxin S6b. 5. In strip preparations with intact endothelium, the pD2 of ET-l increased to 9.04 =/- 0.16 (vs.8.50 +/- 0.07 in rings), and BQ-123 (1 microM) caused a rightward shift of the ET-l induced concentration response curve (pKB 6.62 vs. 5.75 in rings).6. Contractile responses to big-ET-1 of endothelium-intact coronary artery segments were attenuated in the presence of phosphoramidon (100 microM), indicating conversion of big-ET-1 to ET-1 within the coronary artery segment.7. The present study indicates that ET-1 and sarafotoxin S6b contract the human isolated coronary artery via different receptors, which can probably be best characterized as subtypes of the ETA receptor.Furthermore, it is demonstrated that the type of preparation (ring or strip) may affect the potency of ET-1 as an agonist and of BQ-123 as an antagonist.

Topics: Adolescent; Adult; Azepines; Child; Child, Preschool; Coronary Vessels; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Endothelium, Vascular; Female; Glycopeptides; Humans; In Vitro Techniques; Indoles; Infant; Male; Middle Aged; Muscle Contraction; Muscle, Smooth, Vascular; Peptides, Cyclic; Protease Inhibitors; Protein Precursors; Receptors, Endothelin; Vasoconstrictor Agents; Viper Venoms

1. Endothelin-1 (21-amino acids) and pre-pro endothelin-1 (39-amino acids) produced a concentration-dependent increase in perfusion pressure when infused through the renal artery of rabbit isolated perfused kidney. Addition of phosphoramidon to the medium caused a potentiation in the vasoconstrictor response to endothelin-1 and greatly, but not completely, inhibited vasoconstriction induced by pre-pro-endothelin-1. 2. Addition of indomethacin to the medium did not alter the vasoconstrictor effects of the peptides. 3. Methylene blue in the medium caused a highly significant potentiation in the vasoconstrictor response to endothelin-1. 4. A short-term infusion of endothelin-1 through the renal artery elicited a decrease in urine flow which returned to control levels after perfusing the kidney with Krebs buffer, but prolonged infusion of the peptide produced an irreversible increase in urine flow. 5. Phosphoramidon, methylene blue and indomethacin failed to alter the effect of endothelin-1 on urine flow. 6. From these results it was concluded that phosphoramidon-sensitive endothelin-converting enzyme, probably localized in microvasculature of the kidney, can convert pre-pro-endothelin-1 to endothelin-1 which is responsible for the vasoconstrictor effect of pre-pro-endothelin-1 in addition to its possible direct vasoconstrictor effect on kidney vasculature. Moreover, the endothelin-1 degradating enzyme in kidney should be a phosphoramidon-sensitive metalloproteinase(s). The results also indicated the release of EDRF but not prostanoids by endothelin-peptides in the rabbit isolated perfused kidney.

Topics: Animals; Endothelin-1; Endothelins; Female; Glycopeptides; In Vitro Techniques; Infusions, Intra-Arterial; Kidney; Kinetics; Male; Nitric Oxide; Perfusion; Protein Precursors; Rabbits

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and non-isotopic measurement of endothelin-1 and big endothelin-1. The sensitivities of detection were 0.5 and 0.3 fmol/well, with ED50 values of 13 and 12 fmol/well for the endothelin-1 and big endothelin-1 assays, respectively. Each assay is highly selective for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only recognizes the 21-amino acid biologically active peptide. 2. The two assays were used to measure the effects of two classes of protease inhibitor on the basal release of enothelin-1 and big endothelin-1 from cultured first-passage human umbilical vein endothelial cells. 3. The secretion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P < 0.05), with a concomitant increase in the secreted levels of big endothelin-1 (P < 0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significant decrease in the secretion of endothelin (P < 0.05). However, unlike phosphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affected the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antimalarials; Cells, Cultured; Endothelin-1; Endothelins; Endothelium; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Glycopeptides; Humans; Metalloendopeptidases; Pepstatins; Protein Precursors; Umbilical Veins

Intravenous (i.v.) or intrarenal arterial (i.r.a.) injection of big endothelin-1 (big ET-1) (1.6 nmol/kg) in anesthetized rats produced a significant increase in mean arterial pressure (MAP), a decrease in renal blood flow (RBF) and an increase in renal vascular resistance (RVR). Although the pressor responses to big ET-1 of i.v. and i.r.a. injection were not significantly different, i.r.a. injection of big ET-1 caused a significantly greater reduction in RBF than that seen with i.v. injection of big ET-1. The effects of i.r.a. injection of the peptide on MAP and RBF were markedly suppressed by phosphoramidon. Big ET-1 caused dose-dependent pressor effects in isolated perfused rat kidney and these pressor effects were significantly suppressed by phosphoramidon. Thus, renal vasculature possesses a phosphoramidon-sensitive endothelin converting enzyme, which may play an important role for the renal hemodynamic regulation.

Topics: Animals; Blood Pressure; Endothelin-1; Endothelins; Glycopeptides; In Vitro Techniques; Injections, Intravenous; Kidney; Male; Protein Precursors; Rats; Rats, Sprague-Dawley; Renal Circulation; Vascular Resistance; Vasoconstriction

Endothelin-1 (ET-1) is known to stimulate glycogenolysis in perfused rat livers and isolated rat hepatocytes. To determine the potential action of endothelin's precursor, big endothelin-1 (big ET-1), isolated rat livers were perfused with big ET-1 in a non-recirculating system. Thereby, big ET-1 (10 nM) induced a maximally three-fold increase (P < 0.01 vs. basal values) in hepatic glucose production at 60 min, which was almost completely abolished by concomitant infusion of 50 microM phosphoramidon, a sensitive inhibitor of the enzymatic cleavage of big ET-1 to ET-1. The corresponding incremental release of glucose by big ET-1 was 20.9-fold higher in the absence of phosphoramidon than in its presence (P < 0.01). In contrast, phosphoramidon did not inhibit hepatic glucose production induced by ET-1 (1 nM), glucagon (1 nM), and phenylephrine (5 microM). Glycogenolytic responses to 1 nM ET-1 (P < 0.01), but not to 1 nM glucagon (n.s.) were blocked by indomethacin (100 microM), indicating that prostaglandin release by non-parenchymal cells is at least in part involved in the hepatic ET-1 action. In conclusion, big ET-1 induces hepatic glucose release, which is suggested to depend on intrahepatic conversion of big ET-1 to ET-1 by a phosphoramidon-sensitive pathway.

Topics: Animals; Endothelin-1; Endothelins; Glucagon; Glucose; Glycopeptides; In Vitro Techniques; Indomethacin; Liver; Male; Neprilysin; Phenylephrine; Protein Precursors; Rats; Rats, Sprague-Dawley

We have cloned cDNA encoding bovine endothelin converting enzyme (ECE). The predicted amino acid sequence of bovine ECE consisted of 758 amino acid residues. Northern blot analysis revealed that ECE mRNA was abundantly expressed in lung. Co-expression of the cloned cDNA of bovine ECE with human preproET-1 cDNA in CHO-K1 cells resulted in the production of mature ET-1 and this production was inhibited by phosphoramidon.

Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Base Sequence; Blotting, Northern; Cattle; CHO Cells; Cloning, Molecular; Cricetinae; DNA, Complementary; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Gene Expression; Glycopeptides; Humans; Lung; Metalloendopeptidases; Molecular Sequence Data; Protein Precursors; Recombinant Proteins; RNA, Messenger; Transfection

The structure activity relationship of phosphoramidon analogues was studied for their ability to reduce the hypertensive effect of exogenous proET-1, probably via inhibition of an endothelin converting enzyme activity (ECE). Results concerning in vivo ECE and in vitro thermolysin inhibitions were compared. In contrast to the phosphoryl group of phosphoramidon, which was found to be an absolute requirement, the rhamnose moiety was of very little importance for the inhibition of either enzyme. Furthermore, the tryptophan residue of phosphoramidon appeared to be particularly important for the ECE inhibition, whereas thermolysin inhibition seemed to depend greatly on the leucine residue. It is concluded that in vivo ECE and thermolysin differ in the way they recognise phosphoramidon. The existence of an hydrophobic pocket, specific for the recognition of the tryptophan residue of phosphoramidon, could be proposed for ECE.

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Male; Metalloendopeptidases; Neprilysin; Protein Precursors; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Thermolysin

The effects of phosphoramidon, a metalloproteinase inhibitor, on the pressor and renal actions of big-endothelin (BET), the precursor of porcine Endothelin-1 (ET), was studied in rats. In control rats, BET (0.3, 1.0, and 3.0 nmol/kg) elicited a marked increase in mean arterial blood pressure (from 110 +/- 7 to 105 +/- 7, 120 +/- 8, 147 +/- 6 mm Hg, respectively), and a prominent, dose-dependent, diuretic and natriuretic response (fractional sodium excretion (FENa) increased from 0.4 +/- 0.2 to 0.8 +/- 0.2, 3.1 +/- 0.1, and 8.5 +/- 1.7%, respectively). Pretreatment with phosphoramidon (10 mg/kg + 0.25 mg/kg/min) completely abolished the increase in blood pressure induced by BET, but the diuretic-natriuretic effects were only partially inhibited (FENa increased from 2.0 +/- 0.9 to 3.7 +/- 1.5, 3.9 +/- 1.3, and 4.3 +/- 1.2%, respectively, P < 0.05). Rats treated with phosphoramidon only had no natriuresis over time (FENa changed from 1.9 +/- 0.5 to 2.3 +/- 0.3, 1.6 +/- 0.4, 1.7 +/- 0.6 respectively, P--NS). The data suggest that, unlike the vascular type of the enzyme, the renal endothelin converting enzyme is relatively insensitive to phosphoramidon. Further, diuresis and natriuresis can be induced by BET in the absence of any pressor effect.

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Diuresis; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Kidney; Male; Metalloendopeptidases; Natriuresis; Neprilysin; Protein Precursors; Rats; Rats, Sprague-Dawley; Vascular Resistance

We compared the effects of endothelin-1 and its precursor, big endothelin-1, on vascular resistance of a blood-perfused ex vivo stomach segment of chloralose-anesthetized dogs. In separate groups of dogs, endothelin-1 or big endothelin-1 was infused intra-arterially directly to the gastric segment. Endothelin-1 caused statistically significant dose-related increases in gastric vascular resistance at final blood concentrations of 0.15-10 nM. Although each dose was given for only 5 min, endothelin-1 at concentrations > 0.6 nM caused sustained responses with vascular resistance remaining above control values for approximately 45-90 min. In contrast, however, big endothelin-1 caused a small but statistically significant vasoconstriction only at the highest concentration (10 nM). In other experiments, using 15-min peptide infusions, we found that pretreatment with phosphoramidon, an inhibitor of endothelin-converting enzyme, markedly reduced response to big endothelin-1 but not to endothelin-1. Our results demonstrate that endothelin-1, but not big endothelin-1, is a potent vasoconstrictor of the canine gastric microcirculation. In addition, it appears that big endothelin-1 is degraded to endothelin-1 in the stomach by a phosphoramidon-sensitive metalloproteinase.

Topics: Animals; Dogs; Endothelin-1; Endothelins; Glycopeptides; Male; Protein Precursors; Stomach; Vascular Resistance; Vasoconstriction

We examined conversion of Big endothelin-1 (ET-1) to mature ET-1 and pressor action during perfusion of the isolated perfused rat lung with Big ET-1. Big ET-1 caused a concentration-related increase in perfusion pressure and the pressor molar potency of the peptide was fivefold less than that of ET-1. Pressor responses to Big ET-1 were accompanied by an increase in immunoreactive-ET (IR-ET) levels in the perfusate and in the lung tissues. Pretreatment with phosphoramidon (10(-4) M), a metalloproteinase inhibitor, markedly suppressed the pressor action and increment in IR-ET in the tissues. Unexpectedly, the amount of IR-ET in the perfusate during perfusion of Big ET-1 was not influenced by phosphoramidon treatment. On the other hand, chymostatin, an inhibitor of chymotrypsin-like enzymes, effectively suppressed IR-ET levels in the perfusate; however, this enzyme inhibitor was without effect on the pressor action of Big ET-1 or on the increase in IR-ET levels in lung tissues. We tentatively conclude that the phosphoramidon-sensitive conversion of Big ET-T to ET-1 is linked to the pressor action of Big ET-1 in the isolated perfused rat lung. In addition, it seems likely that chymostatin-sensitive conversion of Big ET-1 to ET-1 does not play a major role in the conversion of the precursor to the mature form. We propose that IR-ET present in the tissues rather than that in the perfusate is a better indicator of the functional conversion of Big ET-1 in the rat lung.

Topics: Animals; Aspartic Acid Endopeptidases; Chymotrypsin; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; In Vitro Techniques; Kinetics; Lung; Male; Metalloendopeptidases; Oligopeptides; Perfusion; Protein Precursors; Pulmonary Circulation; Rats; Rats, Sprague-Dawley

To investigate the potential role of endothelin-1 (ET-1) in fetal vasoregulation, we examined in sheep the hemodynamic effects of infusion of big ET-1 (bET-1; precursor of ET-1) on the systemic and pulmonary circulations in chronically catheterized late-gestation fetuses. Thirteen animals [134 +/- 0.5 (SE) days gestation] received systemic infusions of bET-1 (1.5 or 3.0 micrograms/min for 10 min via the superior vena cava), which increased systemic arterial pressure by 5.0 +/- 1.9 (P < 0.01) and 13.9 +/- 1.8 mmHg (P < 0.01), respectively. Pretreatment with 10 mg of phosphoramidon, an ET-1-converting enzyme inhibitor, blocked the hypertensive response to bET-1. Six animals (136 +/- 1.5 days gestation) received intrapulmonary infusion of bET-1 (3.0 micrograms/min for 10 min via the left pulmonary artery), which increased pulmonary arterial pressure by 18.1 +/- 1.5 mmHg (P < 0.01). Three animals (130 +/- 1.5 days gestation) received phosphoramidon (1 mg/min for 10 min via the left pulmonary artery), which had no observed effect on baseline pulmonary vascular tone. We conclude that bET-1 produces systemic and pulmonary hypertension in the late-gestation fetus. Phosphoramidon inhibits bET-1-induced hypertension, suggesting that the fetus possesses ET-1-converting enzyme activity.

Topics: Animals; Endothelin-1; Endothelins; Female; Fetus; Glycopeptides; Hemodynamics; Injections; Protein Precursors; Pulmonary Circulation; Sheep

The effects of platelets on endothelin-1 (ET-1) production were examined by using cultured bovine pulmonary artery endothelial cells (ECs). Platelets (6 x 10(6) to 2 x 10(9) platelets/ml) prepared from rat peripheral arterial blood markedly stimulated immunoreactive (IR)-ET release from ECs into the culture medium, in a time-and platelet number-dependent manner. High-performance liquid chromatography analysis of the culture supernatant following exposure to platelets revealed one major IR-ET component corresponding to the elution position of synthetic ET-1. Northern blot analysis showed that platelets enhanced prepro ET-1 mRNA expression in the ECs. Increased IR-ET release was observed with the supernatant obtained after incubation of platelets, and this increment was significantly inhibited by transforming growth factor-beta 1 neutralizing antibody. Phosphoramidon, an ET converting enzyme inhibitor, significantly decreased the amount of IR-ET accumulating in the culture medium of ECs, incubated with or without platelets, and the decreasing effect of phosphoramidon in the presence of platelets was greater than that in their absence. A similar effectiveness of phosphoramidon was seen when transforming growth factor-beta 1 was used instead of platelets. Thus, platelets appear to stimulate the endothelial production of ET-1 in vitro, probably through a release of transforming growth factor-beta 1. We also suggest that the inhibition of ET converting enzyme by phosphoramidon is more effective in the augmented condition of ET-1 production than in the basal condition.

Topics: Animals; Blood Platelets; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Culture Media; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Neutralization Tests; Protein Precursors; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

The effects of metal chelators on endothelin (ET)-converting enzyme (ECE) activity in vivo were examined. Three compounds, (2,3-dimercapto-1-propanol (DMP), toluene-3,4-dithiol (TDT) and 8-mercaptoquinoline (8-MQ)), which inhibited ECE in in vitro studies, exhibited inhibitory activity towards big ET-1-induced sudden death in mice, while EDTA did not. Similar results were obtained in big ET-1-induced hypertension. Big ET-1-induced hemoconcentration was inhibited by pretreatment with 8-MQ or EDTA but not with DMP or TDT. The elevation of immunoreactive ET-1 (IR-ET-1) in plasma after administration of big ET-1 was inhibited by pretreatment with the three compounds but not by EDTA. On the other hand, no chelator inhibited the elevation of IR-ET-1 in lung tissue after injection of big ET-1. Taking into consideration the in vitro results, more selective chelating activity of the compounds towards Zn2+ rather than Ca2+ and Mg2+ may contribute to the inhibition of big ET-1-induced responses in vivo. The ET-1 formation involved in big ET-1-induced hemoconcentration may have different physiological characteristics from that involved in big ET-1-induced sudden death or hypertension.

Topics: Animals; Aorta, Thoracic; Aspartic Acid Endopeptidases; Blood Pressure; Chelating Agents; Death, Sudden; Dimercaprol; Edetic Acid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hematocrit; Lung; Male; Metalloendopeptidases; Mice; Mice, Inbred ICR; Muscle Contraction; Muscle, Smooth, Vascular; Protein Precursors; Quinolines; Rats; Rats, Sprague-Dawley; Toluene

We compared the pressor response to endothelin-1 (ET-1) with that of big endothelin-1 (big ET-1) in mesenteric arteries, hindquarters and lungs of rats. In these three preparations, both peptides caused a concentration-dependent increase in the perfusion pressure. The ratio of big ET-1 concentration to ET-1 concentration needed for causing the same pressor action is different between organs; i.e., a mesentery >> a hindquarter > or = a lung. Exposure to phosphoramidon, a metalloproteinase inhibitor, significantly suppressed the pressor response to big ET-1, in a similar fashion. This suppression is likely to be due to the inhibition of phosphoramidon-sensitive endothelin converting enzyme, since the inhibitor does not suppress an action of ET-1. Apparently there is a difference in potency for phosphoramidon-sensitive vasoconstriction of big ET-1 between organs and presumably regional differences in the functional phosphoramidon-sensitive conversion of big ET-1 in vasculatures.

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hindlimb; Lung; Male; Mesenteric Artery, Superior; Metalloendopeptidases; Perfusion; Protein Precursors; Rats; Rats, Sprague-Dawley; Vasoconstriction

Like endothelin-1 (ET-1), its immediate human precursor big ET-1 (1-100 nM) increased the rate of spontaneous phasic contractions and caused graded tonic contractions of isolated rat uterus strips. The tonic contraction to big ET-1 (10 nM) was markedly blocked by phosphoramidon (100 microM), which did not modify the response to an equipotent concentration of ET-1 (3 nM). Responses to big-ET-1 (30 nM) were abolished in calcium-free medium, but those to ET-1 (10 nM) were only reduced by this condition. The EC50 of big ET-1 for inducing tonic contraction was only sevenfold greater than that of ET-1, and both peptides produced a maximal response similar to that evoked by KCl 80 mM. ET-3 was much less potent. The selective ETA receptor antagonist BQ-123 (40-600 nM) caused graded rightward shifts of the ET-1 curve without affecting the maximal response, yielding a Schild plot with a slope not different from unity and a pA2 value of 7.76. BQ-123 (100 nM) did not affect contractions induced by oxytocin (5 nM), acetylcholine (3 microM), or bradykinin (0.3 nM), but inhibited responses to both big ET-1 and ET-1. Therefore, the rat uterus contains a phosphoramidon-sensitive, calcium-dependent endothelin-converting enzyme that readily converts big ET-1 into ET-1, which then contracts the myometrium via activation of ETA receptors.

Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Female; Glycopeptides; In Vitro Techniques; Metalloendopeptidases; Molecular Sequence Data; Peptides, Cyclic; Protein Precursors; Rats; Rats, Wistar; Receptors, Endothelin; Uterine Contraction; Uterus

Experiments were designed to investigate the reactivity of vascular smooth muscle to endothelins (ETs) in veins taken from human placentas immediately after delivery. The placental veins were cut into rings and suspended between two stirrups in conventional organ chambers (filled with aerated, modified Krebs-Ringer bicarbonate solution) for isometric recording of tension. ET-1 and ET-3 caused concentration-dependent contractions of the isolated human placental veins. The responses induced by ET-1 were greater than those evoked by ET-3 and were not significantly affected by BQ-123, a selective inhibitor of ETA receptors. Contractions to big ET-1 were obtained in rings both with and without endothelium; they were inhibited by phosphoramidon, an inhibitor of endothelin-converting enzyme. These findings indicate that the conversion of the precursor of ET-1 can occur in human placental veins. The receptors mediating the contraction of human placental veins to endothelins do not belong to the ETA subtype; the response to the peptides is probably mediated in part by an uncharacterized ET-receptor subtype and in part by ETB receptors. The output of big ET-1 in the vascular wall or from surrounding tissues in the placenta could be involved in the regulation of venous tone in this organ.

Topics: Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Female; Glycopeptides; Humans; In Vitro Techniques; Isometric Contraction; Muscle, Smooth, Vascular; Peptides, Cyclic; Placenta; Pregnancy; Prostaglandins; Protein Precursors; Regional Blood Flow

The activity of human big endothelin-1 (bET-1) or endothelin-1 (ET-1) was investigated in the isolated perfused kidney of the rabbit. In some experiments the effluent superfused a rabbit jugular vein (RbJV) or rat colon (RC), and bolus doses of bET-1 or ET-1 were administered either directly over the tissue (OT) or through the kidney (TK). When injected OT, the contractile responses of the RbJV to ET-1 were > or = 100-200 times more than those to bET-1. However, at least 10 times the ET-1 dose given OT was needed TK to produce an equivalent contraction of the RbJV, showing that ET-1 was being inactivated or removed by the kidney. Injections of bET-1 TK produced increases in perfusion pressure of a magnitude 1/25th of those of ET-1. In some experiments administration of bET-1 TK was associated with the release into the perfusate of an ET-1-like factor that contracted the RbJV, but not the RC (used to detect any angiotensin II release), at doses that had little effect when administered OT, suggesting that bET-1 was being activated by the kidney. Phosphoramidon (10 microM) infused TK blocked (92 +/- 1% inhibition) the renal responses to bET-1 and reduced the overflow of ET-1-like material onto the tissues without affecting ET-1-induced renal vasoconstriction. Incubation of bET-1 with rabbit renal cortical microsomes (100 micrograms protein) resulted in the generation of ET-1-like activity, as assessed by bioassay, which was inhibited by phosphoramidon (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biotransformation; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Glycopeptides; Humans; In Vitro Techniques; Kidney; Kidney Cortex; Male; Microsomes; Protein Precursors; Rabbits; Subcellular Fractions

Several studies have shown that phosphoramidon (PHOS) reduces the release of endothelin-1 (ET-1) from cultured endothelial cells. Moreover, the main endothelin-converting enzyme (ECE) activity in these cells is a membrane-bound metallopeptidase that is also inhibited by PHOS. We have investigated further the role of the PHOS-sensitive ECE in the conversion of big ET-1 to ET-1. ET-1 was measured using a radioimmunoassay specific for the C-terminal ET[16-21] sequence. The effect of PHOS on the production of ET-1 from endogenous precursors was determined using cultured bovine aortic endothelial cells (BAECs) and the human endothelial cell line (EA.hy 926). The concentrations of ET-1 accumulating in the medium over 24 h from BAECs were lowered by PHOS (-27% 10 microM, -76% 100 microM). In contrast, with EA.hy 926 cells, the same concentrations of PHOS increased by five- to sixfold the amount of ET-1 present in the medium after 24-h incubation. In other experiments, incubation of big ET-1 (1 microM) with intact BAECs or EA.hy 926 cells resulted in the generation of ET-1, and with both cell types this was inhibited by PHOS (IC50: BAECs = 6.4 microM; EA.hy 926 = 0.26 microM). These results are consistent with both cell types having a PHOS-sensitive ECE that is readily accessible to exogenous big ET-1 and is therefore probably located on the plasma membrane. Furthermore, another intracellular ECE may play a part in the endogenous intracellular formation of ET-1 in EA.hy 926 cells.

Topics: Amino Acid Sequence; Animals; Aorta; Cattle; Cell Line; Cell Membrane; Cells, Cultured; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Molecular Sequence Data; Protein Precursors; Radioimmunoassay

Endothelin-1 (ET-1) is a powerful renal vasoconstrictor peptide that could be implicated in acute renal failure. The aim of this study was to test the effects of the endothelin-converting enzyme (ECE) inhibitor phosphoramidon on pressor responses to ET-1 and its precursor, big ET-1, in isolated perfused rat kidneys and in pithed rats. In Tyrode-perfused rat kidneys, both big ET-1 (0.2-0.4 nmol) and ET-1 (0.01-0.03 nmol) evoked dose-dependent constrictions. Phosphoramidon (10 microM) selectively inhibited the pressor responses to big ET-1 without altering those to ET-1, norepinephrine, angiotensin I (AT-I), or angiotensin II (AT-II). The metalloprotease inhibitor thiorphan, but not the angiotensin-converting enzyme (ACE) inhibitor perindoprilate, also selectively inhibited the renal constrictions caused by big ET-1 but not those induced by ET-1. In vivo, both big ET-1 and ET-1 (0.5-2 nmol/kg) evoked pressor responses that were augmented by indomethacin (15 mg/kg) and L-NNA (1 mg/kg/min). Phosphoramidon selectively inhibited the pressor responses to big ET-1 (ID50: 78 micrograms/kg/min) without affecting those to ET-1, AT-I, or AT-II. These data illustrate that the pressor responses to big ET-1 in the rat, both in vivo and in vitro, are due to its conversion into ET-1 by a phosphoramidon-sensitive ECE. In the rat, phosphoramidon selectively inhibits ECE but not ACE both in vitro and in vivo.

Topics: Angiotensin I; Angiotensin II; Animals; Aspartic Acid Endopeptidases; Blood Pressure; Decerebrate State; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hemodynamics; In Vitro Techniques; Kidney; Male; Metalloendopeptidases; Norepinephrine; Protein Precursors; Rats; Rats, Sprague-Dawley; Rats, Wistar

The pharmacology of human big endothelin-1 (big ET-1) and big ET-3 was compared in five pharmacologic models: perfused rat and guinea pig lungs, perfused rabbit kidney, and in the rat and the guinea pig in vivo (blood pressure monitoring). In these models, big ET-1 consistently induced concentration- or dose-dependent pharmacologic effects sensitive to phosphoramidon (vasopressor or prostanoid-releasing effects). In contrast, big ET-3, dissolved in either phosphate-buffered saline (pH 7.4) or 0.1% acetic acid, was inactive in all the models used in this study. In addition, the activity of big ET-3 was also assessed in the prostatic portion of the rat vas deferens. In this model, although big ET-1 induced a phosphoramidon-sensitive increase of the twitch response of the tissue to electrical stimulation, big ET-3, dissolved either in phosphate-buffered saline or acetic acid, remained inactive. Our results, presented in the above-mentioned models, illustrate the capacity of the phosphoramidon-sensitive endothelin-converting enzyme (ECE) to discriminate between human big ET-1 and big ET-3.

Topics: Animals; Aspartic Acid Endopeptidases; Eicosanoids; Electric Stimulation; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Epoprostenol; Female; Glycopeptides; Guinea Pigs; Humans; In Vitro Techniques; Lung; Male; Metalloendopeptidases; Protein Precursors; Rabbits; Rats; Rats, Wistar; Renal Circulation; Substrate Specificity; Vas Deferens; Vascular Resistance

Intravenous injection of big endothelin-3 (big ET-3, 1-41 amide) at 3 nmol/kg to ganglion-blocked anaesthetized rats produced a long-lasting hypertensive action, but the action was less potent than that seen with the same dose of ET-3-(1-21). The pressor effect induced by big ET-3 was markedly attenuated by pretreatment with phosphoramidon, 5 mg/kg i.v., a dose which had no effect on the hypertensive action induced by ET-3. These results strongly suggest that big ET-3 is converted to mature ET-3 by a phosphoramidon-sensitive metalloproteinase in vivo, in a manner similar to the conversion of big ET-1 to ET-1.

Topics: Anesthesia; Animals; Blood Pressure; Endothelin-1; Endothelins; Ganglionic Blockers; Glycopeptides; Male; Protein Precursors; Rats; Rats, Sprague-Dawley; Thermolysin

1. In the anaesthetized, ganglion-blocked rat, intravenous boluses of endothelin-1, endothelin-2 and endothelin-3 induced a transient hypotensive effect followed by a potent long lasting pressor response (ED50 mmHg: 0.72 +/- 0.05, 1.8 +/- 0.2 and 2.7 +/- 0.3 nmol kg-1, respectively). The maximal effect for the three peptides was of a similar order of magnitude (delta MAP: 84 to 89 mmHg). Neither of these effects was influenced by phosphoramidon or thiorphan (10 mg kg-1, i.v.). 2. Intravenously administered big-endothelin-1 and -2 induced a transient (1-2 min) hypotension followed by a potent long lasting (> 25 min) vasopressor effect (ED50 mmHg: 1.8 +/- 0.2 and 6.7 +/- 0.4 nmol kg-1, respectively), with a similar maximal activity (delta MAP: 85 +/- 4 and 81 +/- 2.4 mmHg, respectively). The onset of the big-endothelin-1 vasopressor effect was more rapid (5-6 min) than that of big-endothelin-2 (10-13 min). Big-endothelin-3 was found to induce only a potent, long lasting (> 35 min) hypertension, with a maximal effect of 75 +/- 4.6 mmHg at 10 nmol kg-1 and an ED50 mmHg of 6.5 +/- 0.4 nmol kg-1. The onset of this effect was much slower (20-25 min) than that of the other proendothelins. Pressor responses induced by big-endothelin-1, -2 and -3 (3, 15 and 10 nmol kg-1, respectively) were markedly reduced (60, 80 and 100%) in the presence of phosphoramidon (10 mg kg-1, i.v.). Thiorphan (10 mg kg-1, i.v.) did not inhibit the effects of big-endothelin-1, -2 and -3. 3. In the electrically stimulated rat vas deferens, endothelin-I and -2 were found to be equipotent enhancers of the twitch response (EC100%: 4.0 +/- 0.4 nm and 7.9 +/- 4.8 nm, respectively), both about 3-4 fold as active as endothelin-3 (EC100%: 19 +/- 2.5 nM). Endothelin-1 and -3 showed a comparable maximalstimulatory effect (Emax: 296 +/- 30 and 262 +/- 24%) while endothelin-2 was less active (Emax: 194 +/- 30%).4. Big-endothelin-l and -2 were potent enhancers of the twitch response too (EC 100,%: 10.0 +/- 2.6 nM and 21.6 +/- 3.2 nM, respectively), with a comparable maximal stimulatory effect (Emax: 254 +/- 22 and 264 +/-24%). Big-endothelin-3 was found to be less potent (EC,100%: 275 +/- 21 nM), but retained a marked potentiating effect (Emax: 200 +/- 38%). Phosphoramidon, but not thiorphan, concentration-dependently(10 and 100 microM) reduced big-endothelin-1 (58 and 86% respectively) and big-endothelin-2 (21 and 56%)-mediated responses. Conversely, the big-endothelin-3 effect was reduced by phosphor

Topics: Animals; Blood Pressure; Dose-Response Relationship, Drug; Electric Stimulation; Endothelin-1; Endothelin-3; Endothelins; Ganglionic Blockers; Glycopeptides; Hemodynamics; In Vitro Techniques; Male; Muscle, Smooth; Neurons; Protein Precursors; Rats; Rats, Sprague-Dawley; Thiorphan; Vas Deferens

The IC50 values of phosphoramidon, CGS 25015, CGS 26129, thiorphan and benazeprilat for inhibition of endothelin converting enzyme partially purified from porcine aortic endothelial cells were 3.5, 18, 58, > 100 and > 100 microM, respectively. A similar rank order of potency was observed for inhibition of the proendothelin-1 (proET-1) -induced pressor response in the rat where phosphoramidon, CGS 25015, CGS 26129, thiorphan and benazeprilat at 30 mg/kg i.v. produced 65, 57, 27, 12, and 0% inhibition, respectively. A slightly different rank order of potency was obtained in the proET-induced contraction of porcine coronary arteries where IC50 values of < 10, 10-30, 10-30, 30-100 and 30-100 microM were exhibited by CGS 25015, CGS 26129, phosphoramidon, thiorphan and benazeprilat, respectively. These data indicate that the endothelin converting enzymes in the three systems studied are similar, except that phosphoramidon is a slightly more potent inhibitor in the in vitro assay and the in vivo pressor test than in the smooth muscle contraction assay.

Topics: Animals; Aspartic Acid Endopeptidases; Benzazepines; Blood Pressure; Coronary Vessels; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; In Vitro Techniques; Male; Metalloendopeptidases; Methionine; Muscle Contraction; Muscle, Smooth, Vascular; Neprilysin; Propionates; Protein Precursors; Rats; Rats, Sprague-Dawley; Swine; Thiorphan

We reported previously that the endothelin converting enzyme (ECE) inhibitor phosphoramidon lowers mean arterial pressure (MAP) when infused in conscious, spontaneously hypertensive rats (SHRs). In this study we determined the dose-response relationship for this action in SHRs and in a high-renin hypertensive model, the renal artery-ligated rat. We also determined whether the ETA receptor antagonist BQ-123 (cyclo [D-Trp-D-Asp-Pro-D-Val-Leu]) might lower MAP in hypertensive rats. Phosphoramidon lowered MAP by 9 +/- 4, 31 +/- 4, and 40 +/- 4 mm Hg after 5 h when infused in SHRs at 10, 20, and 40 mg/kg/h. This lowering of MAP was associated with dose-related inhibition of the pressor response to a bolus intravenous injection of big ET (1-39) at 1 nmol/kg. BQ-123 also lowered MAP in SHRs (by 25 +/- 3 mm Hg), but only at a very high dose (50 mg/kg/h for 5 h). At this dose, BQ-123 blocked the pressor response to a bolus intravenous injection of ET-1 (1 nmol/kg), but the blockade was incomplete. Phosphoramidon infused in conscious, renal hypertensive rats lowered MAP by 31 +/- 9, 46 +/- 8, and 54 +/- 1 mm Hg after 5 h at 10, 20, and 40 mg/kg/h, respectively. This lowering of MAP was associated with blockade of the pressor response to big ET (1-39). BQ-123 did not lower MAP in renal hypertensive rats when infused at 30 mg/kg/h for 5 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Angiotensin I; Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hypertension; Hypertension, Renal; Male; Metalloendopeptidases; Molecular Sequence Data; Neprilysin; Peptides, Cyclic; Protein Precursors; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley

We asked whether or not the endothelium plays a functional role in the conversion of big endothelin-1 to endothelin-1 in the perfused rat mesenteric artery. In endothelium-denuded preparations, big endothelin-1 produced a much more potent pressor effect than in intact preparations. Phosphoramidon suppressed the big endothelin-1-induced pressor action without affecting the action of endothelin-1, irrespective of the presence or absence of the endothelium. The amounts of immunoreactive-endothelin in the perfusate during perfusion of endothelium-denuded preparations with big endothelin-1 were extremely low compared with those observed in intact preparations and were not significantly suppressed by the metalloproteinase inhibitor, phosphoramidon, in contrast to the case with intact preparations. When synthetic endothelin-1 was perfused in the endothelium-denuded mesentery, the peptide disappeared from the perfusate more rapidly than with intact preparations, suggesting that endothelin-1 generated from big endothelin-1 is effectively trapped by vascular smooth muscle cells in the endothelium-denuded preparation. Our results suggest that the endothelium is not essential for the conversion of big endothelin-1 to endothelin-1, in rat mesenteric artery.

Topics: Animals; Blood Pressure; Carbachol; Cholic Acids; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; In Vitro Techniques; Male; Mesenteric Artery, Superior; Neprilysin; Norepinephrine; Perfusion; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley

The presence of a phosphoramidon-sensitive endothelin-1-converting enzyme was investigated in the rat isolated uterus. Endothelin-1 and its precursor, big-endothelin-1, increased the rate of spontaneous contractions and caused tonic contractions. Responses to big-endothelin-1 had a slower start than those to endothelin-1. The tonic contraction induced by big-endothelin-1 (10 nM) was nearly abolished by phosphoramidon (100 microM), but the response to an equieffective concentration of endothelin-1 (3 nM) was not affected. Big-endothelin-1 (EC50 6.7 nM) was only 7-fold less potent than endothelin-1 (EC50 0.9 nM), whereas endothelin-3 was much less potent (EC50 > 100 nM). The endothelin ETA receptor antagonist, BQ-123 (40, 150 and 600 nM), induced graded rightward shifts of the concentration-response curve for endothelin-1. Schild analysis yielded a straight line with a slope not different from unity, and a pA2 value of 7.76. At 100 nM, BQ-123 specifically blocked responses to both endothelin-1 (3 nM) and big-endothelin-1 (10 nM), without modifying those to oxytocin (5 nM), acetylcholine (3 microM) or bradykinin (0.5 nM). Our results suggest the presence of phosphoramidon-sensitive endothelin-converting enzyme and demonstrate the occurrence of functional endothelin ETA receptors in the rat uterus.

Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Female; Glycopeptides; In Vitro Techniques; Metalloendopeptidases; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Peptides, Cyclic; Protein Precursors; Rats; Rats, Wistar; Receptors, Endothelin; Uterus

1. The profile of haemoconcentration induced by big endothelin-1(big ET-1), a precursor of endothelin-1 (ET-1), was compared with that induced by endothelin-1 in mice. 2. ET-1(1.5 nmol kg-1, i.v.) increased haematocrit in mice, which reached a maximum at 5 min and then returned to the control value within 30 min after the administration, this occurred at the same time as changes in the plasma immunoreactive endothelin-1 and rat atrial natriuretic peptide (rANP)-like activities (IR-ET-1 and IR-rANP, respectively). 3. Big ET-1(2.5-15 nmol kg-1, i.v.) also caused a significant and dose-dependent increase in haematocrit, that lasted over 3 h although elevated plasma IR-ET-1 and IR-rANP had almost been restored to the initial levels within 10 min after big ET-1 injection. 4. A metalloproteinase inhibitor, phosphoramidon (10 mg kg-1, i.v.), which inhibits the activity of endothelin converting enzyme (ECE), delayed the onset of big ET-1-induced haemoconcentration, but failed to alter the maximal value and the duration of the haemoconcentration. 5. Pretreatment with phosphoramidon (10 mg kg-1, i.v.) did not affect the big ET-1-induced change in plasma IR-ET-1, while significant delay of the disappearance of plasma IR-rANP and significant suppression of a sustained increase in tissue IR-ET-1 were observed. 6. These results suggest that ET-1, not in plasma but in tissue, plays an important role in the pathogenesis of big ET-1-induced long-lasting haemoconcentration, in which unknown factors besides rANP are involved.

Topics: Animals; Atrial Natriuretic Factor; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Glycopeptides; Hematocrit; Male; Mice; Mice, Inbred ICR; Protein Precursors

Endothelin-1 (ET-1), a 21-amino acid peptide produced by endothelial cells, results from the cleavage of preproendothelin, generating Big ET-1, which is then cleaved by the ET-converting enzyme (ECE) to form ET-1. Big ET-1, like ET-1, is released by endothelial cells. Big ET-1 is equipotent to ET-1 in vivo, whereas its vasoactive effects are less in vitro. It has been suggested that the effects of Big ET-1 depend on its conversion to ET-1. ET-1 has potent vasoactive effects in the newborn pig pulmonary circulation, however, the effects of Big ET-1 remain unknown. Therefore, we studied the effects of Big ET-1 in isolated perfused lungs from 1- and 7-day-old piglets using the ECE inhibitor, phosphoramidon, and the ETA receptor antagonist, BQ-123Na. The rate of conversion of Big ET-1 to ET-1 was measured using radioimmunoassay. ET-1 (10(-13) to 10(-8) M) produced an initial vasodilation, followed by a dose-dependent potent vasoconstriction (P < 0.001), which was equal at both ages. Big ET-1 (10(-11) to 10(-8) M) also produced a dose-dependent vasoconstriction (P < 0.001). The constrictor effects of Big ET-1 and ET-1 were similar in the 1-day-old, whereas in the 7-day-old, the constrictor effect of Big ET-1 was less than that of ET-1 (P < 0.017).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Animals, Newborn; Endothelin-1; Endothelins; Glycopeptides; In Vitro Techniques; Peptides, Cyclic; Protein Precursors; Pulmonary Circulation; Radioimmunoassay; Swine

1. Inhibitors of neutral endopeptidase EC.3.4.24.11 (NEP) have been shown to attenuate the hypertensive effect of big-endothelin-1 (BET-1) in rats. To determine whether NEP converts BET-1 to endothelin-1 (ET-1), the effect of a recombinant NEP (rNEP) on BET-1 and on ET-1 was assessed in vitro. 2. Incubation of [125I]-ET-1 with 1 microgram ml-1 of rNEP resulted in degradation of the peptide within minutes. Increase in the amount of rNEP to 10 micrograms ml-1 led to total cleavage of [125I]-ET-1 within seconds. 3. Phosphoramidon (10 microM) or SQ-28,603 (100 microM) totally suppressed the degradation of [125I]-ET-1 by rNEP. 4. The degradation of [125I]-BET-1 by either 1 or 10 micrograms ml-1 of rNEP was much slower than that of [125I]-ET-1. Again, both phosphoramidon and SQ 28,603 protected the peptide from degradation. 5. Intact [125I]-ET-1 was not observed when [125I]-BET-1 was incubated with rNEP. 6. These data show that neutral endopeptidase EC.3.4.24.11 is not an endothelin converting enzyme.

Topics: Alanine; Chromatography, High Pressure Liquid; DNA, Complementary; Endothelin-1; Endothelins; Glycopeptides; Humans; In Vitro Techniques; Iodine Radioisotopes; Neprilysin; Protein Precursors; Recombinant Proteins

The endothelin-converting enzyme (ECE) activity present in endothelial cells and rat and human brains was characterized using a selective and rapid bioassay for endothelin-1 (ET-1) or endothelin-3 (ET-3) together with a sensitive enzyme-linked immunosorbent assay. We found that ECE activity was predominantly in the membrane fraction of endothelial cells from which it could be extracted by treatment with detergent. In rat brain tissue, the ECE activity was in the membrane fraction and was not solubilized by detergent treatment. Further dissection of the brain revealed that there was a strong localization of ECE activity in the hypothalamus, midbrain, and medulla oblongata in agreement with earlier observations of ET-like immunoreactivity and binding sites. Experiments with human brain tissue also showed the presence of ECE activity. In conclusion, our studies confirmed the presence of ECE activity within endothelial cells, and showed ECE to be localized in brain tissue in sites consistent with the selective distribution of the ET-1 synthetic pathway. In all tissues studied, the ECE activity was significantly inhibited by phosphoramidon or ethylenediaminetetra-acetate.

Topics: Animals; Aspartic Acid Endopeptidases; Biological Assay; Brain; Cattle; Cell Line; Cells, Cultured; Cyclic GMP; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Glycopeptides; Hypothalamus; Medulla Oblongata; Mesencephalon; Metalloendopeptidases; Neprilysin; Protein Precursors

Using the endothelin-1 (ET-1)-stimulated elevation in cGMP in LLC-PK1 cells as a biological detector system for the conversion of big ET-1 (bET-1) to ET-1, we detected bET-1-converting activities in subcellular fractions from bovine aortic cultured endothelial cells (BAE) and rat brain. Within the particulate fraction of BAE, we detected two activities, at pH 3.4 and pH 5.4-7.4. The latter but not the former activity was inhibited in a concentration-dependent manner by phosphoramidon (approximate IC50, 1 microM) and converted bET-1 to ET-1 at a rate of 0.6 nmol/hr/mg of protein. It could be solubilized from the particulate fraction by detergent treatment. Phosphoramidon-inhibitable converting activity was also detected in the cytosolic fraction of BAE. Within the rat brain, phosphoramidon-inhibitable conversion of bET-1 to ET-1 was detected principally in the cytoskeletal fraction, i.e., that fraction from the membrane that was not solubilized by detergent treatment. These results show the presence of at least two different endothelin-converting enzyme activities in endothelial cells and a third within the rat brain. They also demonstrate the use of LLC-PK1 cells as a rapid assay that permits the sensitive detection and measurement of the formation of biologically active ET-1 from its precursor bET-1.

Topics: Animals; Aorta; Aspartic Acid Endopeptidases; Brain; Cattle; Cells, Cultured; Cyclic GMP; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Epithelial Cells; Epithelium; Glycopeptides; Kidney; Male; Metalloendopeptidases; Protein Precursors; Rats; Rats, Inbred Strains; Subcellular Fractions; Swine

In anaesthetized and ventilated guinea-pigs, i.v. injection of 1 nmol/kg big endothelin-1 (big ET-1) did not evoke significant changes in pulmonary inflation pressure (PIP) and mean arterial blood pressure (MBP), whereas injection of the same dose of endothelin-1 (ET-1) induced marked and rapid bronchoconstrictor and pressor responses. Administered at the dose of 10 nmol/kg, big ET-1 provoked significant increases in PIP and MBP, which developed slowly and were long-lasting as compared to those evoked by ET-1. When big ET-1 was incubated for 45 min at 37 degrees C with alpha-chymotrypsin (2 mU/nmol) or pepsin (1 microgram/nmol) and then injected into guinea-pigs at the dose of 1 nmol/kg, marked bronchoconstrictor and pressor responses were observed, with kinetics similar to those noted after administration of the same dose of ET-1. The magnitude of the alpha-chymotrypsin- or pepsin-treated big ET-1 responses was similar to that induced by ET-1, incubated or not with the enzymes. Injected i.v. at the dose of 5 mg/kg, 5 min before the challenge, phosphoramidon almost totally inhibited the bronchoconstrictor and pressor responses induced by 10 nmol/kg big ET-1, whereas thiorphan (5 mg/kg) partially reduced the increase in PIP and exerted a minimal effect on the changes in MBP. Administered at the dose of 20 mg/kg per os, 1 h before i.v. administration of 10 nmol/kg big ET-1, enalapril maleate and captopril did not significantly alter the bronchoconstriction and the hypertensive response evoked by the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Bronchi; Chymotrypsin; Endothelin-1; Endothelins; Glycopeptides; Guinea Pigs; Lung; Male; Neprilysin; Pepsin A; Protein Precursors; Sensitivity and Specificity; Thiorphan

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.

Topics: Animals; Aorta; Aspartic Acid Endopeptidases; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Lung; Metalloendopeptidases; Protein Precursors; Rats; Swine; Time Factors

The effects of big endothelin-1 and endothelin-1 on vascular reactivity were compared in isolated rabbit saphenous artery and vein. The contractile potency of endothelin-1 was three times higher in the vein than in the artery. In contrast, big endothelin-1 was two times more effective in the artery. Phosphoramidon (100 microM), a metalloproteinase inhibitor, antagonised the contractile action of big endothelin-1 in the artery but not in the vein. These data suggest that the biosynthetic pathway leading to the formation of endothelin differs in arterial and venous vessels.

Topics: Animals; Arteries; Endothelin-1; Endothelins; Glycopeptides; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Neprilysin; Protein Precursors; Rabbits; Saphenous Vein; Vasoconstriction

1. Changes in vascular permeability following intravenous injections of human big-endothelin-1 (big-ET-1) and endothelin-1 (ET-1) were measured by extravasation of Evans blue dye (EB, 20 mg kg-1) in selected tissues. 2. A low dose of big-ET-1 (40 pmol kg-1) failed to alter vascular permeability but a dose of 400 pmol kg-1 increased EB extravasation in the trachea, upper and lower bronchi, and lung parenchyma by 55 to 69% (P < 0.05). Vascular permeability was also enhanced in the liver, spleen, kidney, heart, and diaphragm by 20, 14, 41, 25, and 67%, respectively (P < 0.05). 3. Upon injection of ET-1 (400 pmol kg-1), EB extravasation increased in the upper and lower bronchi, lung parenchyma, liver, pancreas, kidney, heart, and diaphragm. 4. Administration of ET-1 and big-ET-1 was not associated with significant systemic responses. 5. Pretreatment with phosphoramidon (PA) blocked the response to big-ET-1 in all tissues examined but this inhibitor failed to alter the response to ET-1. 6. We conclude from these results that the dose-dependent increase in vascular permeability induced by big-ET-1 in various tissues follows its conversion to ET-1 by the endothelin converting enzyme, a PA-sensitive process.

Topics: Animals; Blood Pressure; Bronchi; Capillary Permeability; Coronary Vessels; Endothelin-1; Endothelins; Evans Blue; Glycopeptides; Injections, Intravenous; Kidney; Lung; Male; Protein Precursors; Rats; Rats, Wistar; Spleen; Trachea

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.

Topics: Adenocarcinoma; Aspartic Acid Endopeptidases; Chromatography, High Pressure Liquid; Cross Reactions; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Humans; Kidney Neoplasms; Metalloendopeptidases; Protein Precursors; Radioimmunoassay; Tumor Cells, Cultured

The effect of endothelin-1 (ET-1) and big ET-1 on coronary flow and contractile function was determined in isolated nonischemic and ischemic rat hearts. Both ET-1 (IC50 = 12 pMol) and big ET-1 (IC50 = 2 nMol) reduced coronary flow in a concentration-dependent manner, although ET-1 was > 100-fold more potent. Both compounds decreased contractility, an effect which was lost when coronary flow was held constant, indicating that ET-1 and big ET-1 decrease contractility secondary to reducing coronary flow. Mechanical reduction in coronary flow to levels equivalent to those seen for ET-1 or big ET-1 caused similar reductions in contractility. Both 30 pMol ET-1 and 10 nMol big ET-1 pretreatment significantly reduced the time to contracture in globally ischemic rat hearts, suggesting a proischemic effect. Phosphoramidon (100 microM, endothelin-converting enzyme inhibitor) and BQ-123 (0.3 microM, ETA receptor antagonist) abolished the preischemic increase in coronary perfusion pressure induced by big ET-1 as well as its proischemic effect, whereas only BQ-123 abolished the cardiac effect of ET-1. Neither phosphoramidon nor BQ-123 had an effect on severity of ischemia when given alone. Phosphoramidon was also given i.v. to rats subjected to coronary occlusion and reperfusion and was found to significantly reduce infarct size 24 hr postischemia. Thus, in isolated rat hearts, big ET-1 appears to be converted to ET-1 and is a potent coronary constrictor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Coronary Circulation; Coronary Vessels; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Glycopeptides; In Vitro Techniques; Male; Myocardial Contraction; Myocardial Ischemia; Peptides, Cyclic; Protein Precursors; Rats; Rats, Sprague-Dawley

To investigate vascular responses to the endothelin-1 (ET-1) precursor, human big endothelin 1-38 (big ET), in the peripheral vascular bed of the cat.. These studies were designed to investigate the hypothesis that bit ET is converted to an active peptide with properties similar to ET-1.. Hindquarters vascular bed of the cat under conditions of controlled bloodflow; changes in perfusion pressure reflect changes in vascular resistance.. Fifty-four adult mongrel cats.. Big ET, ET-1, the peptidases chymotrypsin, pepsin and cathepsin-D, and the metalloprotease inhibitor phosphoramidon.. Intra-arterial injections of big ET induced a slow-developing and sustained increase in hindquarters perfusion pressure which could be blocked by phosphoramidon. ET-1 (0.3 nmol), administered as a slow infusion over a 10-min period, produced a slowly developing increase in hindquarters perfusion pressure in a manner similar to that observed in response to injection of big ET. A bolus injection of ET-1 produced a biphasic response characterized by a transient decrease in pressure followed by an increase which was significantly greater in magnitude and more rapid in onset than the pressor response to big ET (0.3 nmol). After incubation of big ET with chymotrypsin, pepsin and cathepsin-D (each 5% weight/weight) for 30 mins at 37 degrees C, injection of activated big ET produced a biphasic response characteristic of the response to ET-1 with an initial transient decrease in pressure followed by a secondary increase in hindquarters perfusion pressure.. Big ET produces a phosphoramidon-sensitive pressor response which is similar to that produced by an infusion of ET-1. These data suggest that chymotrypsin, pepsin and cathepsin-D can convert big ET to an active peptide which elicits a biphasic response similar to that produced by ET-1.

Topics: Animals; Blood Pressure; Blood Vessels; Cathepsin D; Cats; Chymotrypsin; Dose-Response Relationship, Drug; Drug Combinations; Endothelin-1; Endothelins; Glycopeptides; Hindlimb; Infusions, Intra-Arterial; Injections, Intra-Arterial; Neprilysin; Pepsin A; Protein Precursors; Regional Blood Flow

Experiments were conducted to determine the importance of the carbohydrate moiety of phosphoramidon in the inhibition of the pressor response to big endothelin-1 in anesthetized rats. Big endothelin-1 produced a 42% increase in mean arterial pressure which was nearly abolished by co-infusion of phosphoramidon. Similarly, when an analog of phosphoramidon lacking the rhamnose group (phosphoryl-leu-trp-OH) was co-infused, a significant attenuation of the pressor response was observed. These findings indicate that the rhamnose moiety of phosphoramidon is not responsible for the distinguishing this compound as an inhibitor of the response to big endothelin-1 in the rat.

Topics: Animals; Anti-Bacterial Agents; Aspartic Acid Endopeptidases; Blood Pressure; Dipeptides; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Male; Metalloendopeptidases; Protein Precursors; Rats; Rats, Inbred Strains; Rhamnose; Structure-Activity Relationship

Incubation of big endothelin-1 (bET-1) with protein derived from the detergent-extracted 100,000 g pellet prepared from human brain tissue resulted in the formation of endothelin-1 (ET-1) at a rate of 90 fmol mg-1 protein min-1. This formation was inhibited in a concentration-dependent manner by either phosphoramidon or EDTA, with half-maximal inhibitory concentrations of 2 and 20 microM, respectively. No conversion of big endothelin-3 (bET-3) to endothelin-3 (ET-3) was detected under the same conditions. These results show the presence in the human brain of a metalloprotease-like enzymatic activity which selectively converts bET-1 and ET-1. Together with earlier reports of mRNA for ET-1 this suggests the presence of the entire synthetic pathway for ET-1 in human brain.

Topics: Brain; Dose-Response Relationship, Drug; Edetic Acid; Endothelin-1; Endothelins; Glycopeptides; Humans; Metalloendopeptidases; Neprilysin; Protein Precursors

Previous studies of endothelin-1 (ET) synthesis have shown that some cultured endothelial cells secrete an intermediate product, big-endothelin-1 (bigET), suggesting that the processing of secreted bigET to ET may be physiologically significant. In this study, two pertinent ET converting enzyme activities, mast cell chymase I (EC 3.4.21.39) and a phosphoramidon-sensitive, neutral metalloprotease, were identified in a rat lung particulate fraction. We perfused rat lungs with bigET and chymostatin or phosphoramidon to study the relevance of these two proteases to the processing of extracellular bigET in vivo. Addition of compound 48/80 (a compound which activates mast cells, causing degranulation and release of chymase) to the perfusion buffer greatly increased hydrolysis of exogenously added bigET to ET. ET formation was inhibited completely by 32 microM chymostatin, whereas inhibition by 50 microM phosphoramidon was incomplete and variable. Perfusate histamine levels were used to monitor the extent of mast cell degranulation, and inhibition of ET production by phosphoramidon was attributed to inhibition of degranulation, per se. There was a direct correlation between perfusate ET and histamine levels in both control and phosphoramidon-treated (but not chymostatin-treated) lungs. Our results suggest that chymase from lung mast cells is capable of physiologically relevant extracellular processing by bigET to ET in the perfused rat lung.

Topics: Animals; Aspartic Acid Endopeptidases; Chymases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hydrogen-Ion Concentration; Lung; Mast Cells; Metalloendopeptidases; Oligopeptides; Perfusion; Protein Precursors; Rats; Rats, Inbred Strains; Serine Endopeptidases

The lethal activity of big endothelin-1 (bET-1) was examined in mice and compared with endothelin-1 (ET-1). Like ET-1, intravenous administration of bET-1 produced sudden death with an approximate LD50 value at 21.0 nmol/kg, higher than that of ET-1 (3.8 nmol/kg). At doses above the respective LD90 value, the latency to death was much longer in bET-1-treated mice with sustained elevation of plasma immunoreactive ET-1 (IR-ET-1). A metalloproteinase inhibitor, phosphoramidon, although failing to inhibit sudden death induced by ET-1, suppressed bET-1-induced lethality and elevation of plasma IR-ET-1 probably due to an inhibition of the enzymatic conversion of bET-1 to ET-1.

Topics: Animals; Chromatography, High Pressure Liquid; Death, Sudden; Drug Antagonism; Endothelin-1; Endothelins; Glycopeptides; Immunoenzyme Techniques; Male; Metalloendopeptidases; Mice; Mice, Inbred ICR; Protein Precursors

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.

Topics: Animals; Aorta; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Neprilysin; Protein Precursors; Radioimmunoassay; Swine

We examined the effects of endothelin-1 (ET-1) on pulmonary hemodynamic and transvascular fluid filtration and the conversion of big endothelin-1 (big ET-1), a precursor of ET-1, in isolated perfused rabbit lungs at constant vascular and airway pressures. Furthermore we examined whether ET-1 contributes to cyclooxygenase metabolism. The perfusate flow decreased significantly after bolus administration of 1 or 0.1 nmol of ET-1. Lung weight did not increase throughout the experimental period. Big ET-1- (1 nmol) induced decrease in the flow was slow in developing, although the maximum response was comparable to that induced by the same dose of ET-1. The concentration of bit ET-1 in the perfusate progressively decreased, while that of ET-1 increased in a time-dependent manner. Phosphoramidon, an inhibitor of metalloproteinase, suppressed the pressor effect of big ET-1 (P less than 0.01) and the increase in the concentration of ET-1 in the perfusate (P less than 0.05). The present findings provide the first evidence suggesting that the potent vasocontractile effect of big ET-1 in pulmonary circulation can be attributed to the production of ET-1 by the conversion from big ET-1 in the vascular bed. ET-1-induced perfusate flow changes were not affected by indomethacin, and the concentration of 6-ketoprostaglandin F1 alpha, a metabolite of prostacyclin, did not increase after ET-1 administration.

Topics: Animals; Blood Pressure; Endothelin-1; Endothelins; Glycopeptides; In Vitro Techniques; Lung; Metalloendopeptidases; Methoxamine; Organ Size; Perfusion; Prostaglandin-Endoperoxide Synthases; Protein Precursors; Pulmonary Circulation; Rabbits

The potent vasoconstrictor endothelin 1 (ET-1) is thought to arise from the proteolytic processing of big endothelin 1 (Big ET) by a unique endothelin-converting enzyme, possibly a metalloprotease. Experiments were conducted to determine the effects of Big ET on cardiovascular and renal functions during inhibition of metalloprotease activity in vivo. Intravenous infusion of Big ET (0.1 nmol.kg-1.min-1) in anesthetized euvolemic rats produced a significant increase in mean arterial pressure (MAP; 39 +/- 8%) and a decrease in effective renal plasma flow (ERPF; -39 +/- 2%), whereas glomerular filtration rate (GFR) remained unchanged (-8 +/- 8%). Simultaneous intravenous infusion of phosphoramidon (0.25 mg.kg-1.min-1), an inhibitor of metalloprotease activity including neutral endopeptidase EC 3.4.24.11 (NEP), completely prevented these effects of Big ET. Thiorphan (0.1 mg.kg-1.min-1), also an inhibitor of NEP, had absolutely no effect on either the renal or cardiovascular response to Big ET. Similarly, the response to Big ET was unaffected by infusion of enalaprilat (0.1 mg.kg-1.min-1), an inhibitor of the angiotensin-converting enzyme, which is also a metalloprotease. To determine whether the effect of phosphoramidon was due to antagonism of ET-1, an identical series of experiments was performed using ET-1 infusion (0.02 nmol.kg-1.min-1). Although the increase in MAP (24 +/- 5%) produced by ET-1 was less than that observed for the given dose of Big ET, the renal vasoconstriction was much more severe; the smaller peptide changed ERPF and GFR by -66 +/- 7 and -54 +/- 9%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Diuresis; Enalaprilat; Endothelin-1; Endothelins; Glomerular Filtration Rate; Glycopeptides; Hemodynamics; Infusions, Intravenous; Kidney; Male; Metalloendopeptidases; Natriuresis; Potassium; Protein Precursors; Rats; Rats, Inbred Strains; Renal Circulation; Thiorphan

Both the soluble and membrane fractions prepared from cultured bovine endothelial cells (ECs) possessed the converting activities to metabolize big endothelin-1 (big ET-1) to endothelin-1 (ET-1) at neutral pH. Metal chelators inhibited the activities of both fractions, whereas phosphoramidon, a metalloprotease inhibitor, strongly inhibited only the activity of the membrane fraction. Phosphoramidon reduced the secretion of ET-1 and concomitantly enhanced the release of big ET-1 from cultured ECs. The incubations of big ET-1, big ET-2, and big ET-3 with cultured ECs resulted in their conversions to mature ETs. Phosphoramidon also abolished these conversions. These results indicate that vascular endothelium can convert not only endogenous big ET-1 but also exogenous big ET isopeptides to their mature ETs through a phosphoramidon-sensitive neutral metalloprotease.

Topics: Animals; Aspartic Acid Endopeptidases; Cattle; Cells, Cultured; Chelating Agents; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium; Glycopeptides; Hydrogen-Ion Concentration; Metalloendopeptidases; Protease Inhibitors; Protein Precursors

Human big endothelin (big ET) and endothelin-1 (ET-1) induce similar increases in left ventricular systolic pressure in the anesthetized rabbit. Unlike ET-1, human big ET does not induce an initial transient hypotension. Human big ET (3 nmol/kg) inhibits ADP-induced platelet aggregation ex vivo by 60% whereas ET-1 at 1 nmol/kg inhibits platelet aggregation by more than 80%. The C-terminal fragment, big ET[22-38] (3 nmol/kg), has no antiaggregatory properties. Inhibition of ex vivo platelet aggregation by human big ET and ET-1 was not seen in rabbits pretreated with indomethacin (5 mg/kg). Human big ET (10(-7) M) or ET-1 (2.5 x 10(-9)-10(-8) M) induced the release of prostacyclin (PGI2) from rabbit, guinea pig, and rat lungs. Phosphoramidon (50 microM, infused 45 min prior to and during administration of peptides) inhibited the prostanoid-releasing properties of human big ET without affecting the release induced by ET-1. Intravascular administration of human big ET (1 nmol/kg) significantly increased the circulating levels of immunoreactive ET-1 (ir-ET-1) for 30 min whereas administration of ET-1 at the same concentration increased the plasma level of ir-ET-1 for 5 min only. Our results suggest that human big ET is converted to ET-1 in the rabbit in vivo. We further suggest that to induce the release of prostanoids in perfused lungs, human big ET needs to be converted to ET-1 by a phosphoramidon-sensitive endothelin-converting enzyme (ECE).

Topics: Adenosine Diphosphate; Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Epoprostenol; Glycopeptides; Guinea Pigs; Humans; In Vitro Techniques; Lung; Male; Metalloendopeptidases; Platelet Aggregation Inhibitors; Protein Precursors; Rabbits; Rats

We investigated endothelin (ET)-converting enzyme and its localization in the vasculature. The membrane and cytosol fractions of cultured endothelial cells of bovine carotid artery contain phosphoramidon-sensitive ET-converting enzymes, and their molecular weights are about 100 and 540 kDa, respectively. The specific conversion of big ET-1 by these enzymes proceeds at pH 7.0 +/- 0.5, and it is inhibited by EDTA, o-phenanthroline, and phosphoramidon. Big ET-3 is converted by the membrane enzyme at a rate about one-tenth that of big ET-1, but it is not converted by the cytosol enzyme. Big ET-1 (but not ET-1)-induced hypertension in rats was remarkably suppressed by pretreatment with phosphoramidon, and big ET-1 (but not ET-1)-induced contraction of isolated coronary arteries, either with or without the endothelium, was substantially suppressed by phosphoramidon. These results suggest an essential role of phosphoramidon-sensitive enzyme(s) in the vascular conversion of big ET-1, and the existence of such enzymes also in nonendothelial cells. We found three converting enzymes operating at different optimal pH values in noncultured vascular smooth muscle cells; two pepstatin-sensitive, cytosolic acid proteinases and a phosphoramidon-sensitive neutral enzyme(s) in the membrane and cytosol. All of these findings strongly suggest the importance of phosphoramidon-sensitive neutral enzymes in the vascular conversion of big ET-1.

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Carotid Arteries; Cattle; Cell Membrane; Cells, Cultured; Cytosol; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; In Vitro Techniques; Metalloendopeptidases; Muscle Contraction; Muscle, Smooth, Vascular; Protein Precursors; Rats; Swine

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate big endothelin (big ET) by a putative endothelin-converting enzyme (ECE) that cleaves the 39-mer at the Trp21-Val22 bond. Because big ET has less than 1% of the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of big ET. Big ET injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET to ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were ineffective at blocking the pressor response to big ET. However, the metalloprotease inhibitors phosphoramidon and thiorphan both dose-dependently inhibited the pressor response to big ET, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on blood pressure when administered alone as an i.v. bolus to the ganglion-blocked anesthetized rat. However, phosphoramidon infused intravenously at 20 mg/kg/h for 4 h lowered the mean arterial pressure (MAP) in conscious spontaneously hypertensive rats (SHRs) whereas kelatorphan at the same dose did not. Our results suggest that ECE is a novel metalloprotease and that ECE inhibitors could have therapeutic potential for the treatment of hypertension.

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hypertension; Male; Metalloendopeptidases; Protease Inhibitors; Protein Precursors; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY

In pentobarbital-anesthetized rats endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3), and mouse ET-2 (mET-2), in contrast to human big ET-1 (h-big ET), administered as i.v. bolus injections (0.25 nmol/kg i.v.) produced rapidly appearing and short-lasting blood pressure decreases. This effect was markedly inhibited (80-100%) after an 8-min i.v. infusion (0.1 nmol/kg/min over 10 min) of any of the ET studied, but not by h-big ET, the precursor of ET-1. Similarly, in pithed rats given a 10 min i.v. infusion of an equipressor dose (0.1 nmol/kg/min) of ET-1 or h-big ET, the hypotensive effects of ET-1 were entirely blocked only in the group of animals pretreated with ET-1. In pithed rats, ET-1 (0.25 nmol/kg i.v.) and h-big ET (0.5 nmol/kg i.v.) produced equivalent maximal pressor responses and the same pattern of increase in systemic, hindquarter, and renal vascular resistance. However, ET-1 was three times more potent than h-big ET as a vasoconstrictor of the mesenteric bed. Also the pressor response to h-big ET, but not ET-1 (0.25 nmol/kg i.v.), was markedly inhibited by the metalloprotease inhibitor phosphoramidon (5 mg/kg i.v.). These results indicate that the hypotensive effects of ET isopeptides have a common mechanism because they elicit cross tachyphylaxis, although h-big ET did not inhibit the decrease in blood pressure produced by ET-1. A possible explanation for this finding is that h-big ET has intrinsic pressor activity but does not have affinity for receptors mediating the vasodepressor effects of ET isoforms. Alternatively, h-big ET is converted into ET-1 too slowly to yield biophase concentrations of ET-1 necessary for lowering blood pressure and developing tachyphylaxis to ET-isoform-induced hypotension. Finally, if the pressor effects of h-big ET are mediated by ET-1 formation, phosphoramidon can be considered as an inhibitor of the endothelin-converting enzyme.

Topics: Animals; Blood Pressure; Decerebrate State; Endothelin-1; Endothelins; Glycopeptides; Male; Protein Precursors; Rats; Regional Blood Flow; Tachyphylaxis

Since the discovery of endothelin-1 (ET-1), its involvement in cerebral vasospasm after subarachnoid hemorrhage (SAH) has been suspected. We performed various experiments, first to demonstrate the presence of ET in both patients and dogs with SAH, and second to examine the effects of ET synthesis inhibition in experimental vasospasm. Here we report that ET was present in both plasma and cerebrospinal fluid (CSF) in SAH, but did not correlate with vasospasm. However, ET was locally expressed in the vascular endothelium in vasospasm. Several therapeutic approaches causing the inhibition of ET synthesis were effective in preventing the development of vasospasm. Such approaches utilized drugs that inhibited RNA and DNA synthesis. Among them, actinomycin D treatment was most effective. We also utilized phosphoramidon, a recently found conversion inhibitor of big ET to ET. However, this product failed to ameliorate the development of vasospasm. Therefore, although we cannot yet conclude that ET is the main cause of cerebral vasospasm, it may, at least, act as one of the modifying factors in cerebral vasospasm.

Topics: Animals; Antineoplastic Agents; Basilar Artery; DNA; Dogs; Endothelin-1; Endothelins; Glycopeptides; Immunohistochemistry; In Vitro Techniques; Ischemic Attack, Transient; Protein Precursors; RNA, Messenger; Subarachnoid Hemorrhage; Transcription, Genetic

When big endothelin-1 (big ET-1, 1-39) was incubated with the membrane fraction obtained from cultured endothelial cells (ECs) at pH 7.0 for 6 h, the immunoreactive (ir) ET in the reaction mixture was markedly increased. Phosphoramidon, a metalloproteinase inhibitor, as well as metal chelators specifically suppressed the above increase. Using reverse-phase high-performance liquid chromatography, ir-ET was confirmed to be ET-1[1-21]. In addition, we noted that the alterations in ET-1 correlated with those in the C-terminal fragment (CTF, 22-39) of big ET-1. When cultured ECs were incubated with phosphoramidon, time-dependent secretion of ET-1 and CTF from the cells was markedly suppressed. In contrast, the secretion of big ET-1 was increased by phosphoramidon. Thiorphan, a specific inhibitor of neutral endopeptidase 24.11, was without effect on the secretion of ET-related peptides. Moreover, phosphoramidon potently inhibited the hypertensive effect of big ET-1 without affecting the ET-1-induced hypertension in anesthetized rats. From these findings, it seems reasonable to consider that phosphoramidon-sensitive and membrane-bound metalloproteinase, which is not a neutral endopeptidase 24.11, is the most plausible candidate for big ET-1-converting enzyme in vivo.

Topics: Animals; Aorta; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Endothelium; Glycopeptides; Male; Membranes; Metalloendopeptidases; Protein Precursors; Rats; Rats, Inbred Strains

Endothelin-1 (ET-1) enhanced field stimulation-evoked (0.1 Hz), nerve-mediated contractions of the prostatic portion of the rat vas deferens. The human precursor of ET-1, big-endothelin (1-38) (big-ET-1) was only two-fold less potent than ET-1 (pD2 values: 7.30 and 7.49, respectively). The threshold concentrations necessary to elicit an increase of the response to electrical stimulation was lower for ET-1 (5 nmol/l) than for big-ET-1 (25 nmol/l). Endothelin-3 (ET-3) also markedly enhanced the response of the tissue to field stimulation with a potency similar to ET-1 (pD2 value: 7.59). In contrast, the precursor of ET-3, big-endothelin (1-41) (big-ET-3), was inactive at concentrations up to 0.5 mumol/l. Treatment of the preparations with phosphoramidon (50 mumol/l) markedly reduced the twitch enhancement by big-ET-1 without affecting the response to ET-1. Our results suggest the presence of a specific phosphoramidon-sensitive endothelin-converting enzyme which converts big-ET-1 to ET-1 in the rat vas deferens.

Topics: Animals; Aspartic Acid Endopeptidases; Electric Stimulation; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Humans; In Vitro Techniques; Male; Metalloendopeptidases; Muscle Contraction; Protein Precursors; Rats; Rats, Inbred Strains; Vas Deferens

1. In anaesthetized rats, an i.v. injection of endothelin-1 (0.25 nmol kg-1) evoked a rapidly appearing (maximal effect within 15 s) and short lasting (3 min) fall in blood pressure with tachyphylaxis occurring so that it was reduced by 50% by the last of 4 injections given 10 min apart. This property was also shared by endothelin-2, endothelin-3 and vasoactive intestinal contractor (VIC). 2. Cross tachyphylaxis between the isopeptides occurred. However, under the same experimental conditions the hypotensive effects of acetylcholine, adenosine, atrial natriuretic peptide (ANP) and substance P were reproducible and not modified in animals in which endothelin-1 no longer lowered blood pressure. Thus, the mechanism of the hypotensive action of endothelin peptides is different from that of acetylcholine, adenosine, ANP, and substance P. 3. In pithed rats, endothelin-1 (0.25 nmol kg-1) and its precursor human proendothelin (h-proendothelin) (0.5 nmol kg-1) induced pressor responses of a similar magnitude, which for h-proendothelin (up to 5.0 nmol kg-1) were not preceded by a hypotensive phase. The pressor effects of endothelin-1, like those of vasopressin, were reproducible upon repeated i.v. injections. 4. Rats given a 10 min infusion (0.1 nmol kg-1 min-1) of endothelin-1 showed no hypotensive response to an i.v. bolus injection of endothelin-1, whereas animals pretreated with an equipressor infusion of h-proendothelin did not develop tachyphylaxis to endothelin-1. 5. In pitched rats, endothelin-1, at a dose inducing the same maximal increase in blood pressure as h-proendothelin, was approximately 3 fold more potent as a mesenteric vasoconstrictor than h-proendothelin. These results suggest that if h-proendothelin is processed to endothelin-1, this transformation is not uniform throughout the vascular system. 6. The pressor response of h-proendothelin in pithed rats was dose-dependently inhibited by phosphoramidon (2.5-5.0mgkg '). However, this compound did not antagonize the effects of endothelin-1(0.25 nmol kg- ) or those of h-proendothelin (0.5 nmol kg- ) once developed. 7. Although some of these results may suggest that h-proendothelin does not undergo in vivo conversion to endothelin-1, the results obtained with phosphoramidon suggest that h-proendothelin is converted into endothelin-1. Therefore, the amount of endothelin-1 so produced can elicit pressor responses or regional vasoconstriction, but is insufficient to lower blood pressure and to inhibit en

Topics: Anesthesia; Animals; Blood Pressure; Carotid Arteries; Decerebrate State; Endothelin-1; Endothelins; Glycopeptides; Male; Nephrectomy; Protein Precursors; Rats; Rats, Inbred Strains; Regional Blood Flow; Tachyphylaxis; Vasopressins

1. Human big-endothelin-1 (big-ET-1) and endothelin-1 (ET-1) are equipotent as pressor agents and produce a significant change in mean arterial blood pressure (MAP) in anaesthetized guinea-pigs (2 nmol kg-1: peak delta MAP: 23 +/- 6 mmHg and 26 +/- 5 mmHg, respectively). 2. Unlike big-ET-1, big-endothelin-3 (big-ET-3) (10 and 20 nmol kg-1) induces no pressor responses whereas endothelin-3 (ET-3) at 2 nmol kg-1 induces a significant increase of blood pressure in anaesthetized guinea-pigs (peak delta MAP: 27 +/- 5 mmHg) with a shorter duration than ET-1 and big-ET-1. 3. Big-ET-1 at concentrations 40 times higher than those required for ET-1 (2.5 nM) releases prostacyclin (PGI2) (maximal release: 2.7 +/- 0.8 ng ml-1; 2.9 +/- 0.9 ng ml-1, respectively) and thromboxane B2 (TxB2) (maximal release: 6.7 +/- 1.3 ng ml-1; 6.8 +/- 1.1 ng ml-1, respectively) from guinea-pig perfused lungs. ET-3 (2.5 nM) is also a potent releaser of PGI2 and TxB2 from the guinea-pig lungs (maximal release: PGI2: 2.4 +/- 1.0 ng ml-1; TxB2: 3.8 +/- 0.6 ng ml-1). Conversely, big-ET-3 (100 nM) does not increase basal release of eicosanoids. 4. Phosphoramidon (50 microM), a metalloprotease inhibitor, markedly reduced the eicosanoid releasing properties of big-ET-1 (n = 4, P less than 0.01) in guinea-pig perfused lungs without affecting the release stimulated by ET-1. 5. Our results suggest that big-ET-1 is converted to ET-1 via a phosphoramidon-sensitive endothelin converting enzyme (ECE) to release eicosanoids. The ECE is present in the guinea-pig pulmonary vasculature. Furthermore, our results suggest that the ECE activity is specific for big-ET-1 and may not convert big-ET-3 to its active metabolite, ET-3.

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Eicosanoids; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Epoprostenol; Glycopeptides; Guinea Pigs; In Vitro Techniques; Male; Metalloendopeptidases; Protein Precursors; Thermolysin; Thromboxane B2

Endothelin(ET)-1 and big ET-1 caused potent and sustained constriction of isolated guinea pig bronchus. The response to ET-1 was enhanced by phosphoramidon in a simple dose-related manner (0.01-1000 microM), while the response to big ET-1 was enhanced at lower doses (0.01-0.1 microM) but was suppressed at higher doses (100-1000 microM) of phosphoramidon. Big ET-1, when given intravenously (i.v.) to anesthetized guinea pigs, increased both bronchopulmonary inflation pressure and mean arterial blood pressure (2.5, 5, 10 nmol/kg i.v.). The pressor response to big ET-1 was attenuated by phosphoramidon dose-relatedly, while the pulmonary response was modified in a complex fashion composed of delayed onset and prolonged duration of action. These results suggest that ET converting as well as degrading enzymes coexist in the airway tissue and both enzymes are sensitive to phosphoramidon, so that phosphoramidon acts bifunctionally to reduce and stimulate the airway responses to big ET-1.

Topics: Animals; Anti-Bacterial Agents; Aspartic Acid Endopeptidases; Bronchi; Bronchoconstriction; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Guinea Pigs; Male; Metalloendopeptidases; Protein Precursors

1. Cardiovascular responses to human proendothelin [1-38], in the absence and presence of phosphoramidon, were studied in conscious Long Evans rats, chronically instrumented for the continuous recording of heart rate, systemic arterial blood pressure and renal, mesenteric and hindquarters blood flows. 2. A dose of 0.1 nmol kg-1 human proendothelin [1-38] caused a slight pressor effect (maximum 5 +/- 2 mmHg), but a clear bradycardia (maximum -29 +/- 7 beats min-1). Renal haemodynamics were unchanged but there was mesenteric vasoconstriction and a vasodilation followed by a vasoconstriction in the hindquarters. 3. A dose of 1.0 nmol kg-1 human proendothelin [1-38] caused a gradual hypertension (maximum 42 +/- 4 mmHg at 10 min) and a profound bradycardia (-149 +/- 10 beats min-1 at 30 min). There were gradual but marked, renal and hindquarters vasoconstrictions, whereas there was a substantial mesenteric vasoconstriction that was relatively rapid in onset. 4. In 2 animals, administration of human proendothelin [1-38] at a dose of 10 nmol kg-1 caused an initial hypotension followed by a rapidly-developing pressor effect; there were renal and mesenteric vasoconstrictions and vasodilatation followed by vasoconstriction in the hindquarters. These changes were very similar to those seen following injection of endothelin-1 (0.1 nmol kg-1). 5. Phosphoramidon (2 mumol kg-1) had no cardiovascular effects itself and it did not affect significantly the pressor or mesenteric vasoconstrictor effects of human proendothelin [1-38], but it reduced the bradycardia and renal and hindquarters vasoconstrictor responses. A higher dose of phosphoramidon (lOnmolkg-') caused significant attenuation of all the responses to human proendothelin [1-38], but a substantial mesenteric vasoconstrictor response still occurred under these conditions. 6 The results are consistent with the involvement of phosphoramidon-sensitive enzyme systems in the conversion of human proendothelin [1-38] to endothelin-1 in vivo. In addition, considering the different patterns of responses to human proendothelin [1-38] in the effector tissues studied (heart, and renal, mesenteric and hindquarters vascular beds), and the differential degrees of inhibition of them by phosphoramidon, it is likely that the effects of human proendothelin [1-38] were due to its local (rather than systemic) conversion to endothelin-1 by processes with differing degrees of susceptibility to phosphoramidon.

Topics: Animals; Doppler Effect; Endothelin-1; Endothelins; Glycopeptides; Hemodynamics; Humans; Kidney; Male; Mesenteric Veins; Protein Precursors; Rats; Rats, Inbred Strains; Regional Blood Flow

Intravenous injections of big endothelin (ET)-1 (700 pmol/kg) in the pig increased arterial plasma levels of ET-1-like immunoreactivity (ET-1-LI) from 11.1 +/- 0.7 pM to 46.3 +/- 6.7 pM in the control situation and from 11.5 +/- 0.4 pM to 58.2 +/- 17 pM in the presence of the neutral endopeptidase inhibitor phosphoramidon (3 mg/kg). Big ET-1 increased splenic vascular resistance by 29% in the control situation. The vasoconstriction evoked by big ET-1 in the spleen was reduced after phosphoramidon treatment whereas the elevation of arterial ET-1-LI was not influenced. Furthermore the splenic vasoconstriction evoked by ET-1 was reduced after phosphoramidon without influencing plasma ET-1-LI. Also in rats the pressor effect of big ET-1 (1 nmol/kg) was inhibited by phosphoramidon (5 mg/kg) whereas the elevation of plasma ET-1 was not influenced. It is concluded that the vasoconstrictor effects of both big ET-1 and ET-1 are inhibited, but the increase in plasma ET-1 is unaffected by phosphoramidon.

Topics: Animals; Endothelin-1; Endothelins; Female; Glycopeptides; Injections, Intravenous; Male; Neprilysin; Protein Precursors; Radioimmunoassay; Rats; Rats, Inbred Strains; Spleen; Swine; Vascular Resistance; Vasoconstriction

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.

Topics: Analysis of Variance; Animals; Blood Pressure; Brain; Cardiovascular System; Endothelin-1; Endothelins; Glycopeptides; Male; Metalloendopeptidases; Protein Precursors; Rats; Rats, Inbred Strains; Renal Circulation

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.

Topics: Amino Acid Sequence; Animals; Aorta; Aspartic Acid Endopeptidases; Cattle; Cytosol; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Metalloendopeptidases; Microsomes; Molecular Sequence Data; Peptides; Protein Precursors; Protein Processing, Post-Translational; Sequence Homology, Nucleic Acid; Substrate Specificity

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate called "big endothelin-1" (big ET-1) by a putative ET-converting enzyme (ECE) that cleaves the 39-mer at the bond between Trp-21 and Val-22. Since big ET-1 has only 1/100-1/150th the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of ET-1. Big ET-1 injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET-1 into ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET-1 in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were all ineffective at blocking the pressor response to big ET-1. However, the metalloprotease inhibitors phosphoramidon and thiorphan dose-dependently inhibited the pressor response to big ET-1, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on mean arterial pressure when administered alone. In a rabbit lung membrane preparation, ECE activity was identified that was blocked by the metalloprotease inhibitors phosphoramidon and 1,10-phenanthroline in a concentration-dependent manner. This enzyme converted big ET-1 to a species of ET that comigrated on HPLC with ET-1 and produced an ET-like contraction in isolated rat aortic rings. Our results suggest that the physiologically relevant ECE is a metalloprotease.

Topics: Animals; Aorta; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Phenanthrolines; Protease Inhibitors; Protein Precursors; Rats; Rats, Inbred Strains; Swine; Vasoconstriction

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.

Topics: Animals; Cattle; Cells, Cultured; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Kinetics; Protease Inhibitors; Protein Precursors; Protein Processing, Post-Translational

The effect of phosphoramidon, a metalloproteinase inhibitor, on the pressor response to big endothelin-1 (big ET-1) in the isolated perfused rat mesenteric artery was examined. Big ET-1 (10(-9)-10(-7) M) caused a concentration-dependent increase in the perfusion pressure. The pressor response was markedly suppressed by phosphoramidon (10(-5) M). The inhibitor did not influence the ET-1 (5 X 10(-11)-10(-8) M)-induced pressor action. Big ET-1 (5 X 10(-8) M)-induced pressor action was accompanied by an increase in immunoreactive (IR)-ET in the perfusate, and this increase was suppressed by the addition of phosphoramidon. IR-ET in the perfusate was confirmed to be ET-1, as determined by reverse-phase HPLC. These findings strongly suggest that phosphoramidon-sensitive metalloproteinase contributes to the conversion of big ET-1 to ET-1, in vivo.

Topics: Animals; Anti-Bacterial Agents; Chromatography, High Pressure Liquid; Endothelin-1; Endothelins; Glycopeptides; In Vitro Techniques; Kinetics; Male; Mesenteric Arteries; Muscle, Smooth, Vascular; Perfusion; Protein Precursors; Protein Processing, Post-Translational; Rats; Rats, Inbred Strains; Vasoconstriction

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.

Topics: Animals; Basilar Artery; Blood Pressure; Dogs; Endothelin-1; Endothelins; Female; Glycopeptides; Injections, Intravenous; Male; Metalloendopeptidases; Protein Precursors; Stereotaxic Techniques

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.

Topics: Animals; Aorta, Thoracic; Cell Membrane; Cells, Cultured; Chromatography, High Pressure Liquid; Cytosol; Edetic Acid; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Hydrogen-Ion Concentration; Metalloendopeptidases; Peptide Fragments; Phenanthrolines; Protein Precursors; Protein Processing, Post-Translational; Swine

Intravenous (i.v.) injection of big endothelin-1 (1-39, 0.05-1.0 nmol/kg) to anaesthetized rats produced a dose-dependent and long-lasting hypertensive effect, the magnitude of which was similar to that evoked by ET-1 (1-21). In animals given phosphoramidon (0.25 mg/kg per min i.v.), a metalloproteinase inhibitor, the hypertensive effect of big endothelin-1 was markedly attenuated. The same dose of phosphoramidon did not influence the endothelin-1-induced hypertensive effect. The possibility that big endothelin-1 is converted to endothelin-1 by a phosphoramidon-sensitive metalloproteinase in vivo warrants further attention.

Topics: Anesthesia; Animals; Blood Pressure; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Glycopeptides; Injections, Intravenous; Male; Metalloendopeptidases; Protein Precursors; Rats; Rats, Inbred Strains

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.

Topics: Animals; Blood Pressure; Coronary Vessels; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Guinea Pigs; Heart Rate; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Protein Precursors; Rats; Rats, Inbred WKY; Respiratory System; Swine; Vasoconstriction