angiotensin-i and hippuryl-histidyl-leucine

Antihypertensive peptides received much interest over the last decade. These peptides are known to be angiotensin converting enzyme (ACE) inhibitors in vitro, but the actual antihypertensive mechanisms in vivo are still unclear. In this research, we used rat aortic rings in organ bath experiments to investigate five potential vascular antihypertensive mechanisms of the dipeptide Val-Tyr. Only one significant effect was observed, namely preincubation of the aorta with Val-Tyr led to a significant shift of the concentration-response curve evoked by angiotensin I (Ang I). Val-Tyr had no effect on the angiotensin II receptor or the alpha-adrenergic receptor. Furthermore, it did not interact with voltage-operated Ca2+ channels, or with nitric oxide production/availability. In conclusion, our results show that Val-Tyr specifically inhibits Ang I-evoked contraction through ACE inhibition and that four other main mechanisms of vascular tone regulation are not affected.

Topics: Acetylcholine; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Aorta; Dipeptides; Dose-Response Relationship, Drug; Free Radical Scavengers; In Vitro Techniques; Lisinopril; Male; Norepinephrine; Oligopeptides; Potassium Chloride; Rats; Rats, Wistar; Vasoconstriction

Angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism-related differences in ACE concentration do not result in differences in angiotensin levels.. To investigate whether this relates to differences in the contribution of the ACE C-domain and N-domain, we quantified, using the C-domain-selective inhibitors quinaprilat and RXPA380, and the N-domain-selective inhibitor RXP407, the contribution of both domains to the metabolism of angiotensin I, bradykinin, the C-domain-selective substrate Mca-BK(1-8), and the N-domain-selective substrate Mca-Ala in serum of IIs, DDs, and 'hyperACE' subjects (i.e., subjects with increased ACE due to enhanced shedding). During incubation with angiotensin I, the highest angiotensin II levels were observed in sera with the highest ACE activity. This confirms that ACE is rate-limiting with regard to angiotensin II generation. C-domain-selective concentrations of quinaprilat fully blocked angiotensin I-II conversion in DDs, whereas additional N-domain blockade was required to fully block conversion in IIs. Both domains contributed to bradykinin hydrolysis in all subjects, and the inhibition profile of RXP407 when using Mca-Ala was identical in IIs and DDs. In contrast, the RXPA380 concentrations required to block C-domain activity when using Mca-BK (1-8) were three-fold higher in IIs than DDs.. The contributions of the C-domain and N-domain differ between DDs and IIs, and RXPA380 is the first inhibitor capable of distinguishing D-allele ACE from I-allele ACE. The lack of angiotensin II accumulation in DDs in vivo is not because of the often quoted concept that ACE is a nonrate-limiting enzyme. It may relate to the fact that in IIs both the N-domain and C- domain generate angiotensin II, whereas in DDs only the C-domain converts angiotensin I.

Topics: Adult; Aged; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Coronary Vessels; Enzyme Activation; Female; Genotype; Humans; In Vitro Techniques; Male; Middle Aged; Oligopeptides; Peptidyl-Dipeptidase A; Phosphinic Acids; Point Mutation; Protein Structure, Tertiary; Sus scrofa; Tetrahydroisoquinolines; Vasoconstrictor Agents; Vasodilator Agents

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.

Topics: Amino Acids; Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Animals; Arthropod Proteins; Brachyura; Captopril; Cations, Divalent; Chromatography, High Pressure Liquid; Edetic Acid; Gills; Half-Life; Invertebrate Hormones; Iodine Radioisotopes; Kinetics; Lisinopril; Male; Metals; Nerve Tissue Proteins; Oligopeptides; Peptidyl-Dipeptidase A; Tissue Distribution

1. The aim of the present study was to determine the effect of nitric oxide (NO) on angiotensin-converting enzyme (ACE) activity. 2. A biochemical study was performed in order to analyse the effect of the NO-donors, SIN-1 and diethylamine/NO (DEA/NO), and of an aqueous solution of nitric oxide on the ACE activity in plasma from 3-month old male Sprague-Dawley rats and on ACE purified from rabbit lung. SIN-1 significantly inhibited the activity of both enzymes in a concentration-dependent way between 1 and 100 microM. DEA/NO inhibited the activity of purified ACE from 0.1 microM to 10 microM and plasma ACE, with a lower potency, between 1 and 100 microM. An aqueous solution of NO (100 and 150 microM) also inhibited significantly the activity of both enzymes. Lineweaver-Burk plots indicated an apparent competitive inhibition of Hip-His-Leu hydrolysis by NO-donors. 3. Modulation of ACE activity by NO was also assessed in the rat carotid artery by comparing contractions elicited by angiotensin I (AI) and AII. Concentration-response curves to both peptides were performed in arteries with endothelium in the presence of the guanylyl cyclase inhibitor, ODQ (10 microM), and the inhibitor of NO formation, L-NAME (0.1 mM). NO, which is still released from endothelium in the presence of 10 microM ODQ, elicited a significant inhibition of AI contractions at low concentrations (1 and 5 nM). In the absence of endothelium, 1 microM SIN-1 plus 10 microM ODQ, as well as 10 microM DEA/NO plus 10 microM ODQ induced a significant inhibition on AI-induced contractions at 1 and 5 nM and at 1-100 nM, respectively. 4. In conclusion, we demonstrated that (i) NO and NO-releasing compounds inhibit ACE activity in a concentration-dependent and competitive way and that (ii) NO release from endothelium physiologically reduces conversion of AI to AII.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Carotid Arteries; Diethylamines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Male; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Oligopeptides; Oxadiazoles; Peptidyl-Dipeptidase A; Quinoxalines; Rabbits; Rats; Rats, Sprague-Dawley

Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. The aim of this study was to purify and characterize the secreted and intracellular ACE forms from mesangial cells in culture.. Medium from Wistar rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. The concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE1, mol. wt 130 000 and ACE2, 60000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. The supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACEInt1, mol. wt 130000 and ACEInt2, 68000) were separated. The purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. The Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5.. The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.

Topics: Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Captopril; Cattle; Cells, Cultured; Chlorides; Chromatography, Gel; Enalaprilat; Glomerular Mesangium; Hydrogen-Ion Concentration; In Vitro Techniques; Isoenzymes; Molecular Weight; Oligopeptides; Peptidyl-Dipeptidase A; Rats; Substrate Specificity; Temperature

Angiotensin-converting enzyme inhibitors have beneficial effects that are presumably mediated by decreased angiotensin II (ANG II) production. In this study, we measure for the first time ANG I and ANG II levels in the interstitial fluid (ISF) space of the heart. ISF and aortic plasma ANG I and II levels were obtained at baseline, during intravenous infusion of ANG I (5 microM, 0.1 ml/min, 60 min), and during ANG I + the angiotensin-converting enzyme inhibitor captopril (cap) (2.5 mM, 0.1 ml/min, 60 min) in six anesthetized open-chested dogs. ISF samples were obtained using microdialysis probes inserted into the left ventricular myocardium (3-4 probes/dog). ANG I increased mean arterial pressure from 102+/-3 (SEM) to 124+/-3 mmHg (P < 0.01); addition of cap decreased MAP to 95+/-3 mmHg (P < 0.01). ANG I infusion increased aortic plasma ANG I and ANG II (pg/ml) (ANG I = 101+/-129 to 370+/-158 pg/ml, P < 0.01; and ANG II = 22+/-40 to 466+/-49, P < 0.01); addition of cap further increased ANG I (1,790+/-158, P < 0.01) and decreased ANG II (33+/-49, P < 0.01). ISF ANG I and ANG II levels (pg/ml) were > 100-fold higher than plasma levels, and did not change from baseline (8,122+/-528 and 6,333+/-677), during ANG I (8,269+/-502 and 6, 139+/-695) or ANG I + cap (8,753+/-502 and 5,884+/-695). The finding of very high ANG I and ANG II levels in the ISF vs. intravascular space that are not affected by IV ANG I or cap suggests that ANG II production and/or degradation in the heart is compartmentalized and mediated by different enzymatic mechanisms in the interstitial and intravascular spaces.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Captopril; Chromatography, High Pressure Liquid; Coronary Vessels; Dogs; Extracellular Space; Heart Rate; Myocardium; Oligopeptides; Peptidyl-Dipeptidase A; Perfusion; Renin-Angiotensin System

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

Exogenous angiotensin I (ANG I) was degraded to mainly des-Asp-ANG I instead of ANG II in the hypothalamic homogenate of the Sprague Dawley (SD), Wistar Kyoto (WKY), left renal artery stenosed hypertensive SD (LRAS), deoxycorticosterone acetate/salt-induced hypertensive SD (DOCA-salt) and spontaneously hypertensive rats (SHR). In the same homogenate, ANG II was degraded to ANG III and ANG III remained unchanged during the first 10 min of incubation. However, all the homogenates were able to catalyse hippuryl-L-histidyl-L-leucine to hippuric acid and the catalysis was completely inhibited by 3 microM captorpil. The data show that the angiotensin converting enzyme present in the hypothalamus when extracted by the normal laboratory procedures is not able to hydrolyse ANG I to ANG II. In addition, the aminopeptidase that degraded ANG I to des-Asp-ANG I was not inhibited by amastatin, bestatin and EDTA, indicating that it is not aminopeptidase A or B. The formation of hippuric acid was significantly higher in the homogenate of the LRAS whilst the SHR and DOCA-salt showed significant higher rate of des-Asp-ANG I formation than in the normotensive control rats.

Topics: Angiotensin I; Animals; Hippurates; Hypertension; Hypothalamus; Oligopeptides; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Tissue Extracts

Selectivity of captopril, enalapril (MK-421), enalaprilat (MK-422), ketoace, and SA-300 for inhibiting kininase II when angiotensin (ANG) I versus bradykinin (BK) is the substrate has been studied in vitro. Potency for inhibiting purified rabbit lung ANG I-converting enzyme (ACE) using a tripeptide substrate with an ANG I-like (hippurylhistidylleucine, HHL) or a BK-like (hippurylphenylalanylarginine, HPA) cleavable dipeptide was determined. Inhibition of ANG I-induced and potentiation of BK-induced contractions of isolated guinea pig ileum strips was measured. For the enzyme assay, the inhibitor concentration which reduced the rate of HHL and HPA hydrolysis 50% (IC50) from the control value was estimated. All tested compounds more potently inhibited hydrolysis of the ANG I-related tripeptide by the purified enzyme. Ketoace, with a selectivity ratio (HPA IC50:HHL IC50) of 23, was the most substrate-dependent inhibitor. For the isolated ileum assay, the inhibitor concentration which augmented the contractile response to BK by 50% (AC50) or inhibited the contractile response to ANG I by 50% (IC50) was calculated. Only enalaprilat retained a selectivity ratio (BK AC50:ANG I IC50) in the guinea pig ileum system greater than one. Ketoace, with a ratio of 0.038, was the least ANG I-selective by this criterion. In vivo selectivity data on captopril seem more in accord with the ileum, rather than the enzyme, results. It was concluded that converting enzyme inhibitors differ in their relative selectivity for inhibiting kininase II reactions using different substrates.

Topics: Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Guinea Pigs; Hydrolysis; In Vitro Techniques; Male; Muscle Contraction; Oligopeptides; Peptidyl-Dipeptidase A; Substrate Specificity

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Angiotensins; Animals; Cross Reactions; Edetic Acid; Enalapril; Leucine; Lisinopril; Oligopeptides; Pepstatins; Peptidyl-Dipeptidase A; Protease Inhibitors; Rabbits; Radioimmunoassay; Renin; Spectrophotometry; Substrate Specificity

The angiotensin converting enzyme (ACE) activity in the plasma collected from normotensive and hypertensive patients was measured chromatographically by two different methods using hippuryl-histidyl-leucine (HHL-ACE) and synthetic angiotensin I (AI-ACE) as substrates. A single peak was observed in the elution profiles of HHL-ACE on both Sephadex G 150 column and DEAE Sephadex A 50 cellulose column chromatographies. A single peak of AI-ACE appeared on the gel-filtration. However, several peaks of AI-ACE were seen on DEAE Sephadex A 50 cellulose column chromatography, suggesting that there are various types of ACE which hydrolyze angiotensin I but not HHL.

Topics: Adult; Angiotensin I; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Female; Humans; Hypertension; Isoenzymes; Male; Oligopeptides; Peptidyl-Dipeptidase A; Renin

An inhibitor of angiotensin I (ANG I) converting enzyme, SA446, reduced the response to ANG I of blood vessels isolated from dogs and monkeys, but did not abolish the response even at high concentrations. The residual action of ANG I in the presence of high concentrations of SA446 could be abolished by (Sar1, Ala8)-ANG II. Vascular strips and crude extracts of vessels and lungs possessed the enzymic activity generating ANG II from ANG I, or hippuric acid from hippuryl-histidyl-leucine (HHL). The HHL-hydrolysing activity of the crude extracts was completely inhibited by SA446 (10(-7) mol/l) and/or Na2-EDTA (10(-3) mol/l). However, the octapeptide generation was not abolished despite the combined treatment with SA446 (5 X 10(-4) mol/l) and Na2-EDTA (5 x 10(-3) mol/l). The residual activity forming ANG II was inhibited by chymostatin and soybean trypsin inhibitor, which however did not affect the HHL-hydrolysis. Combined treatment with SA446 (10(-5) mol/l) and chymostatin (2.5 X 10(-5) mol/l) abolished the vascular action of ANG I but did not alter the action of ANG II. These results strongly suggest that besides the ANG I converting enzyme, another enzyme which generates ANG II is present in vascular tissues and lungs, and may play an important role in the local generation of ANG II, which possibly regulates the regional vascular tone.

Topics: 3-Mercaptopropionic Acid; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Angiotensins; Animals; Blood Vessels; Dogs; Female; In Vitro Techniques; Macaca; Male; Muscle Contraction; Muscle, Smooth, Vascular; Oligopeptides; Sulfhydryl Compounds; Thiazolidines