angiotensin-i and phosphoramidon

PgPepO is a homologue of endothelin-converting enzyme-1 (ECE-1), with which it shares 31% identity. PgPepO was isolated from the periodontal pathogen Porphyromonas gingivalis. Recent studies have suggested a link between periodontal and cardiovascular disease, and several groups have suggested that bacterial and viral infections may contribute to the latter. P. gingivalis possesses the ability to invade, and multiply within, aortic endothelial cells and has been localized to atherosclerotic plaques. PgPepO was expressed and purified to homogeneity and we have begun detailed functional analysis, in terms of substrate preference and inhibitor specificity, in order to provide active-site comparisons with other members of the neprilysin (NEP)/ECE family. PgPepO possesses similar substrate specificity to ECE-1 and has been shown to cleave big endothelin-1 (big ET-1), big ET-2 and big ET-3, converting the substrates into their respective mature endothelin peptides. Substance P, angiotensin I, angiotensin II and neurotensin are all cleaved at multiple sites by PgPepO and the kinetics of these reactions have been compared. The potent vasoconstrictor urotensin II is not hydrolysed by PgPepO. Cleavage of bradykinin by PgPepO occurs at the Pro(7)-Phe(8) bond and is inhibited by the NEP and ECE-1 inhibitor phosphoramidon in a pH-dependent fashion (IC(50) =10 microM at pH 7.0) but not by thiorphan, an NEP-specific inhibitor. PgPepO activity is completely inhibited by EDTA. Characterization of this enzyme is important in elucidating possible links between periodontal pathogens and cardiovascular disorders such as atherosclerosis, and provides an opportunity to gain structural information on a bacterial protein with striking similarity to human ECE-1.

Topics: Angiotensin I; Angiotensin II; Aspartic Acid Endopeptidases; Bacterial Proteins; Bradykinin; Chromatography, High Pressure Liquid; Endothelin-Converting Enzymes; Enzyme Activation; Enzyme Inhibitors; Glycopeptides; Humans; Hydrolysis; Inhibitory Concentration 50; Metalloendopeptidases; Neurotensin; Porphyromonas gingivalis; Substance P; Thiorphan

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 aims of this study were to investigate whether angiotensin II and/or endothelin could contribute to the hypoxic contractile response of isolated rat pulmonary artery. Experiments were performed for 1 h on noradrenaline precontracted arterial rings in hypoxic conditions (95% N2 and 5% CO2). Nicardipine, lisinopril, losartan, phosphoramidon, FR139317 and bosentan were used to block Ca2+ channels, angiotensin I-converting enzyme, AT1 receptors, endothelin-converting enzyme, ETA receptors, and ETA/ETB receptors, respectively. The profile of the hypoxic contractile response was biphasic, displaying, after a short relaxation, a weak and transient contraction (from 2-4 min) and then, before complete relaxation, a slowly developed but sustained contraction (from 14-60 min). Endothelium removal abolished the transient contraction and reduced (-59%) the sustained contraction. Nicardipine did not modify the transient contraction, but concentration-dependently decreased (from -35% to -100%) the sustained contraction (P = 0.024). Lisinopril and losartan did not affect the response (P = 0.418 and P = 0.973, respectively). Bosentan did not modify the transient contraction, but concentration-dependently decreased (from -14% to -71%) the sustained contraction (P = 0.016), whereas phosphoramidon and FR139317 did not affect the response (P = 0.830 and P = 0.806, respectively).. In rat, (i) both phases of the hypoxic contractile response are endothelium-dependent and independent of angiotensin II; (ii) the transient contraction does not depend on endothelin; (iii) the sustained contraction, which involves calcium influx, appears partly dependent on mature endothelin released from storage granules by stimulating ETB receptors.

Topics: Angiotensin I; Angiotensin II; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Azepines; Bosentan; Calcium; Calcium Channel Blockers; Endothelin Receptor Antagonists; Endothelins; Endothelium, Vascular; Extracellular Space; Glycopeptides; Hypoxia; In Vitro Techniques; Indoles; Lisinopril; Losartan; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nicardipine; Pulmonary Artery; Rats; Rats, Wistar; Sulfonamides

Extracts of head parts prepared from the leech Theromyzon tessulatum hydrolyse the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Gly-His bond of benzoyl-Gly-His-Leu. The metabolism of benzoyl-Gly-His-Leu was completely inhibited by captopril, consistent with an angiotensin-converting enzyme activity. Such an enzyme has recently been isolated from T. tessulatum. However, the enkephalin hydrolysis by captopril (100 microM) was inhibited to a maximum of 70%. The residual activity hydrolyzing enkephalin was inhibited by phosphoramidon, consistent with the presence of endopeptidase-24.11, a mammalian enzyme implicated in the metabolism of neuropeptides. This enzyme was isolated using four steps of purification including gel-permeation and anion-exchange chromatographies followed by reverse-phase HPLC. This neuropeptide endopeptidase (of approximate molecular mass 45 kDa) hydrolyses, at pH 7 and 37 degrees C, both the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Phe8-His9 bond of angiotensin I. Cleavage of [D-Ala2, Leu5]enkephalin yields, respectively, the Tyr-D-Ala-Gly and Phe-Leu peptides with a specific activity of 29 nmol Tyr-D-Ala-Gly.min-1.mg protein-1 (Km 95 microM). The hydrolysis of angiotensin I yields angiotensin II and the dipeptide His-Leu with a specific activity of 1.2 nmol angiotensin min-1.mg protein-1 (Km 330 microM). The metabolism of these peptides was totally inhibited by phosphoramidon. This study therefore provides biochemical evidence for neuropeptide-degrading endopeptidases in leeches.

Topics: Amino Acid Sequence; Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Animals; Binding Sites; Captopril; Endopeptidases; Enkephalin, Leucine-2-Alanine; Glycopeptides; Hydrolysis; Kinetics; Leeches; Molecular Sequence Data; Molecular Weight; Neuropeptides; Oligopeptides; Protease Inhibitors; Substrate Specificity

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

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

The aspartic protease, cathepsin E, has been shown to specifically cleave big endothelin (big ET-1) at the Trp21-Val22 bond to produce endothelin (ET-1) and the corresponding C-terminal fragment. To determine whether cathepsin E is a physiologically relevant endothelin converting enzyme (ECE), three novel and potent inhibitors of cathepsin E were administered to conscious rats prior to a pressor challenge with big ET-1. One of the inhibitors of cathepsin E, SQ 32,056 (3 mg/kg i.v.), blocked the big ET-1 response. However, this dose of SQ 32,056 also blocked the pressor response to ET-1. Phosphoramidon specifically inhibited the Big ET-1 pressor response. These results suggest that ECE is not cathepsin E.

Topics: Amino Acid Sequence; Angiotensin I; Animals; Blood Pressure; Cathepsin E; Cathepsins; Endothelins; Glycopeptides; Humans; Male; Molecular Sequence Data; Molecular Structure; Norepinephrine; Oligopeptides; Rats; Rats, Inbred Strains; Reference Values