Effects of intradermal bradykinin after inhibition of angiotensin converting enzyme.

Inhibitors of angiotensin converting enzyme may cause angio-oedema. To see if this might be due to potentiation of the tissue effects of bradykinin the thickness of weals raised by intradermal injection of saline or 1, 3, or 10 micrograms bradykinin was measured before and three times after single doses of captopril, enalapril, or placebo. The mean thickness increased with increasing doses of bradykinin. It did not change with time after the administration of placebo or captopril but increased from 0.61 mm before enalapril to 1.12 mm two and a half hours and 1.06 mm five hours after enalapril was given. Five subjects flushed when given bradykinin after captopril and four after enalapril, but none flushed when given bradykinin after placebo. It is concluded that angiotensin converting enzyme inhibitors potentiate the effects of intradermal bradykinin in vivo and that this may partially explain why they cause angio-oedema in susceptible patients.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

A cardiac specific analog of angiotensin I potentiated by converting enzyme inhibition

[1-Sarcosine, 7-Alanine] angiotensin I [( 1-Sar, 7-Ala] AI) and closely related analogs were tested for inotropic activity in the isolated cat heart, and for pressor activity in the intact conscious sheep both before and during converting enzyme inhibition (CEI). [1-Sar, 7-Ala] AI exhibited potent inotropic activity but was only weakly pressor. [1-Sar] AI, [1-Sar, 5-Val] AI, [1-Sar, 7-alpha MeAla] AI [1-Sar, 5-Val, 7-NMeAla] AI and [1-Sar, 5-Val, 7-Sar] were all potent agonists in both preparations. The action of [1-Sar, 7-Ala] AI was potentiated by CEI in both the isolated heart and the intact sheep. The activity of the remaining analogs was either partially or completely blocked by CEI. The activity of all analogs was inhibited by AII receptor blockade. These data indicate that the nature of the substitution in position 7 determines the affinity of the analog for converting enzyme. The [7-Ala] substitution appears to decrease the effect of the analog upon vascular receptors.     

The angiotensin I converting enzyme.

The angiotensin I converting enzyme (kininase II; peptidyl dipeptidase; EC3.4.15.1) has a dual function: it converts angiotensin I to angiotensin II and it inactivates bradykinin. Lung, kidney, guinea pig plasma and testicles are among the richest sources of the enzyme. Vascular endothelial cells and bursh borders of renal proximal tubular cells contain high concentrations of the enzyme. The availability of synthetic peptide inhibitors was a great help in establishing the function of converting enzyme in normal and pathological conditions.     

Procyanidin structure defines the extent and specificity of angiotensin I converting enzyme inhibition

Recent reports have shown a decrease in blood pressure associated with the consumption of flavanol-containing foods. However, the mechanism behind this effect is not yet known. Previously we demonstrated that the flavanol epicatechin and its related oligomers, the procyanidins, inhibit angiotensin I converting enzyme (ACE) activity in vitro. In this study, we further characterized epicatechin monomer, dimer, tetramer and hexamer ACE inhibitory effect, by performing fluorescence quenching and kinetic assays, using angiotensin I as substrate. Assessment of ACE activity in cultured human umbilical vein endothelial cells (HUVEC) indicated that the tetramer was the most active inhibitor decreasing the formation of angiotensin II by 52% (P<0.001). When ACE activity was measured using isolated rabbit lung ACE, dimer, tetramer and hexamer inhibited angiotensin II production at IC(50) values of 97.0, 4.4, and 8.2 microM, respectively. The quenching of ACE tryptophan fluorescence was assayed to evaluate the molecular interaction between ACE and procyanidins. The hexamer was the most active quencher decreasing ACE fluorescence by 56%, followed by the tetramer and the dimer, decreasing ACE fluorescence by 37% and 36%, respectively. ACE activity was evaluated in the presence of different concentrations of the ACE activator chloride ion (Cl(-)). Increased Cl(-) concentrations reduced IC(50) values for the dimer and tetramer. Finally, ACE inhibition was determined in the presence of different albumin concentrations. The presence of albumin did not reverse the ACE inhibition by dimer and tetramer, but decreased hexamer inhibition by 65%. In summary, the inhibitory effect of procyanidins on ACE and the extent of this inhibition were largely dependent on procyanidin structure. ACE inhibition by procyanidins in vivo might provide a mechanism to explain the benefits of flavonoid consumption on cardiovascular diseases.     

Novel substrates for angiotensin I converting enzyme

Homogenous human angiotensin converting enzyme (EC 3.4.15.1) cleaves dipeptides from the C-terminus of substrates containing a free carboxyl group. In this study we demonstrate that peptides containing a C-terminal nitrobenzylamine are also cleaved by the enzyme. The hydrolysis of these substrates is inhibited by the specific converting enzyme inhibitors captopril and MK421 as well as by anti-converting enzyme antibody. Sodium chloride accelerates the rate of hydrolysis forty-fold. The product of the reaction, an amino acid nitrobenzylamide, was identified by thin layer chromatography and high performance liquid chromatography. These results suggest that the carboxyl group is not an absolute requirement for substrate hydrolysis.     

Structure and activity of angiotensin I converting enzyme inhibitory peptides derived from Alaskan pollack skin

Angiotensin I that converts the enzyme (ACE) inhibitory peptide, Gly-Pro-Leu, previously purified and identified from the Alaskan pollack skin gelatin hydrolysate, were synthesized. In addition, the peptides Gly-Leu-Pro, Leu- Gly-Pro, Leu-Pro-Gly, Pro-Gly-Leu, Pro-Leu-Gly, Gly- Pro, and Pro-Leu, which consisted of glycine, proline, and leucine, were synthesized by the solid-phase method. The IC50 values of each tripeptide. namely Leu-Gly-Pro, Gly- Leu-Pro, Gly-Pro-Leu, Pro-Leu-Gly, Leu-Pro-Gly, and Pro-Gly-Leu. were 0.72, 1.62, 2.65, 4.74, 5.73, and 13.93 microM, respectively. The ACE inhibitory activity of these tripeptides was higher than that of dipeptides, such as Gly- Pro and Pro-Leu with IC50 values of 252.6 and 337.3 microM, respectively. Among the tripeptides, Leu-Gly-Pro and Gly- Leu-Pro had higher inhibitory activity than Gly-Pro-Leu that was isolated from the Alaskan pollack skin gelatin hydrolysate. Among the different types of tripeptides that were examined, the highest ACE inhibitory activity was observed for Leu-Gly-Pro. It had the leucine residue at the N-terminal and proline residue at the C-terminal.     

Modulation of metabolic control by angiotensin converting enzyme (ACE) inhibition

Angiotensin converting enzyme (ACE) inhibitors are a widely used intervention for blood pressure control, and are particularly beneficial in hypertensive type 2 diabetic subjects with insulin resistance. The hemodynamic effects of ACE inhibitors are associated with enhanced levels of the vasodilator bradykinin and decreased production of the vasoconstrictor and growth factor angiotensin II (ATII). In insulin-resistant conditions, ACE inhibitors can also enhance whole-body glucose disposal and glucose transport activity in skeletal muscle. This review will focus on the metabolic consequences of ACE inhibition in insulin resistance. At the cellular level, ACE inhibitors acutely enhance glucose uptake in insulin-resistant skeletal muscle via two mechanisms. One mechanism involves the action of bradykinin, acting through bradykinin B(2) receptors, to increase nitric oxide (NO) production and ultimately enhance glucose transport. A second mechanism involves diminution of the inhibitory effects of ATII, acting through AT(1) receptors, on the skeletal muscle glucose transport system. The acute actions of ACE inhibitors on skeletal muscle glucose transport are associated with upregulation of insulin signaling, including enhanced IRS-1 tyrosine phosphorylation and phosphatidylinositol-3-kinase activity, and ultimately with increased cell-surface GLUT-4 glucose transporter protein. Chronic administration of ACE inhibitors or AT(1) antagonists to insulin-resistant rodents can increase protein expression of GLUT-4 in skeletal muscle and myocardium. These data support the concept that ACE inhibitors can beneficially modulate glucose control in insulin-resistant states, possibly through a NO-dependent effect of bradykinin and/or antagonism of ATII action on skeletal muscle.     

Inhibition of canine lung angiotensin converting enzyme by ACTH and structurally related peptides

Human ACTH and structurally related peptides, such as ACTH 7–38, ACTH 4–11, ACTH 1–10 and ACTH 18–39, noncompetitively inhibited the activity of angiotensin I converting enzyme (dipeptidyl carboxypeptidase; E.C. 3.4.15.1) in the preparation from canine lung. The Ki values were 1.5 μM and 0.54 μM for ACTH and ACTH 7–38, respectively, using[¹⁴C] -Hip-his-leu as the substrate. These results suggest that ACTH and ACTH 7–38 are potent inhibitors of angiotensin I converting enzyme without being substrate for the enzyme.     

Novel mode of action of angiotensin I converting enzyme inhibitors: direct activation of bradykinin B1 receptor

Angiotensin I converting enzyme (kininase II; ACE) inhibitors are important therapeutic agents widely used for treatment in cardiovascular and renal diseases. They inhibit angiotensin II release and bradykinin inactivation; these actions do not explain completely the clinical benefits. We found that enalaprilat and other ACE inhibitors in nanomolar concentrations activate human bradykinin B(1) receptors directly in the absence of ACE and the B(1) agonist des-Arg(10)-Lys(1)-bradykinin. These inhibitors activate at the Zn(2+)-binding consensus sequence HEXXH (195-199) in B(1), which is present also in ACE but not in the B(2) receptor. Activation elevates [Ca(2+)](i) and releases NO from endothelial or transfected cells expressing the B(1) receptor but is blocked by Ca-EDTA, a B(1) receptor antagonist, the synthetic undecapeptide sequence (192-202) of B(1), and the mutagenesis of His(195) to Ala(195). Except for the B(1) antagonist, these agents and manipulations did not block activation by a peptide ligand. Thus, Zn(2+) is essential for B(1) receptor activation by ACE inhibitors at the zinc-binding consensus sequence. Ischemia or cytokines induce abundant B(1) receptor expression. B(1) receptor activation by ACE inhibitors, a novel mode of action reported here first, can contribute to their therapeutic effects by releasing NO in the heart and to some side effects.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Improving efficiency of an angiotensin converting enzyme inhibitory peptide as multifunctional peptides

Bioactive peptides have been defined as specific protein fragments that have numerous biological activities. The aim of this study was to introduce three multifunctional peptides. Hence, we used rabbit lung angiotensin converting enzyme (ACE) inhibitor peptide AFKDEDTEEVPFR to prepare two analogous peptides KDEDTEEVP and KDEDTEEVH. ACE inhibitory, antioxidant, and antimicrobial activities of three synthetic peptides were investigated. Among the three peptides, KDEDTEEVP exhibited the highest ACE inhibitory activity with IC₅₀ value of 69.63 ± 2.51 μM. Furthermore, the results of fluorescence spectroscopy and molecular modeling showed that KDEDTEEVP had more affinity to ACE than other peptides. The peptide of KDEDTEEVH showed the strongest antioxidant scavenging capacity on DPPH radicals (EC₅₀ = 135 ± 9.62 μM), hydroxyl radicals (EC₅₀ = 144 ± 8.73 μM), and ABTS radicals (EC₅₀ = 62 ± 4.52%). Moreover, it showed the highest activity in iron-chelating test (EC₅₀ = 226 ± 14.13 μM) and could also effectively inhibit the peroxidation of linoleic acid. The antimicrobial activity results showed that KDEDTEEVH had higher efficiency against Gram-positive than Gram-negative bacteria with MIC values of higher than 205 ± 10.75 μM. Although there was not a direct correlation between ACE inhibitor and antioxidant activity for analogous peptides, both analogous peptides exhibited more efficiency than the mother peptide. Thus, they can be considered as multifunctional peptides and would be beneficial ingredient to be used in food and drug industry.     

Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Total retro-inverso (TRI) analogues of bradykinin (BK), the B₂a-selective kinin antagonist d-Arg⁰[Hyp₃,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹,Val⁵,Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B₂a bradykinin receptor with affinities (K_d) ranging from 64 M to 4 M. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than agonists. Three TRI peptidomimetics of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Summary Total retro-inverso (TRI) analogues of bradykinin (BK), the B_2a -selective kinin antagonistd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹, Val⁵, Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B_2a bradykinin receptor with affinities (K _d ) ranging from 64 μM to 4 μM. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than ‘agonists’. Three TRI peptidomimetics ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.