Omapatrilat: a new tool for understanding metabolism of bradykinin at the endothelium level.

As a kallikrein-kinin system has been evidenced at the endothelium level, bradykinin released locally could be responsible, at least in part, for the vasodilatory effect of omapatrilat. To objectivate this potential role of bradykinin, it is important to understand the respective role of angiotensin-converting enzyme and neutral endopeptidase in the metabolism of this vasodilatory peptide, and also to define the influence of different pathophysiologic factors on the respective role of these metallopeptidases in this metabolism.

The metabolism of bradykinin: a cornerstone for the understanding of the cardiovascular effects of metallopeptidase inhibitors.

The development of a sensitive and specific analytical approach for the quantification of bradykinin (BK) has enabled the identification of five metallopeptidases primarily responsible for the metabolism of BK, including angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11. Several metabolic studies have shown that the metallopeptidases that participate in BK metabolism vary by location (eg, plasma, endothelium or cardiomyocytes) and by pathology (eg, diabetes, myocardial infarction or left ventricular hypertrophy). These studies advance understanding of the role of endogenous BK in the cardiovascular effects of ACE inhibitors and other metallopeptidase inhibitors.

Comparison of a vasopeptidase inhibitor with neutral endopeptidase and angiotensin-converting enzyme inhibitors on bradykinin metabolism in the rat coronary bed.

The in vitro effects of omapatrilat, a dual vasopeptidase inhibitor that simultaneously inhibits neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), on exogenous bradykinin metabolism after a single passage through the coronary bed were compared with that of a NEP inhibitor (retrothiorphan, 25 nM), an ACE inhibitor (enalaprilat, 130 nM), and omapatrilat (25 nM). Bradykinin and inhibitors were infused into isolated Langendorff rat hearts perfused at 1 ml/min followed by reperfusion at 10 ml/min. Residual bradykinin was quantified in the coronary effluent by enzyme-linked immunosorbent assay to calculate bradykinin recovery and its kinetic parameters (Vmax/Km). Bradykinin degradation rate at 1 ml/min was 4.56 +/- 0.39 1/min per gram without inhibitors and was significantly reduced to 2.57 +/- 0.19 1/min per gram in the presence of enalaprilat, to 2.97 +/- 0.38 1/min per gram with retrothiorphan, to 1.82 +/- 0.17 1/min per gram with both enalaprilat and retrothiorphan, and to 1.14 +/- 0.35 1/min per gram with omapatrilat. In a second set of experiments, the effect of a 14-day treatment of rats with either ACE inhibitors (enalapril, quinapril, and ramipril), a NEP inhibitor (candoxatril), or omapatrilat on exogenous bradykinin metabolism was studied in Langendorff perfused hearts isolated from these long-term treated rats. In untreated rats, bradykinin degradation at a coronary perfusion of 1 ml/min was 4.35 +/- 0.41 1/min per gram. This value was reduced by 30% for the NEP inhibitor, by 50% for all ACE inhibitors, and by 75% for omapatrilat. All inhibitors administered either short term or long term significantly reduced bradykinin degradation during a single passage through the coronary bed. However, omapatrilat administration resulted in the greatest protection from bradykinin breakdown than ACE or NEP inhibitors alone.

Metabolism of bradykinin by the rat coronary vascular bed.

OBJECTIVE: To study the metabolism of bradykinin (BK) after a single passage through the coronary bed in isolated Langendorff rat hearts. METHODS: BK was infused into the aortic flow line to obtain a final concentration of 10 nM, and the coronary, effluent was collected to quantify BK and des-Arg9-BK by competitive enzyme immunoassay. The nature of immunoreactive material was confirmed by immunograms after HPLC separation. The experiments were performed with hearts perfused at either one of the following coronary flow rates: 1, 5 or 10 ml/min. RESULTS: BK recovery without inhibitors was 86.3 +/- 2.9, 60.8 +/- 6.3, and 29.6 +/- 6.8% at 10, 5, and 1 ml/min, respectively. The Vmax/Km ratios at these coronary flow rates were 2.19 +/- 0.72, 4.81 +/- 0.64, and 2.59 +/- 0.33 min-1 g-1), respectively. The angiotensin-converting enzyme (ACE) inhibitor, enalaprilat (130 nM), reduced BK degradation at all flow rates. Inhibition of neutral endopeptidase with retrothiorphan (25 nM) had no effect on BK degradation. However, the combined treatment with enalapril and retrothiorphan reduced BK degradation to lower values than enalaprilat alone. The effect of enzyme inhibitors on BK recovery was inversely related to coronary flow: inhibiting BK degradation markedly increased BK recovery at 1 ml/min, but had no effect at 10 ml/min. The kininase I metabolite of BK, des-Arg9-BK, could not be detected under these experimental conditions. CONCLUSIONS: ACE is the major enzyme responsible for BK degradation during a single passage through the coronary bed. Neutral endopeptidase contributes to BK degradation only when ACE activity is impaired. The effect of enzyme inhibitors on the coronary concentration of BK is highly dependent on coronary flow rate.

Comparative antioxidant and cardioprotective effects of ceruloplasmin, superoxide dismutase and albumin.

The antioxidant effects of ceruloplasmin (CAS 9031-37-2) against oxygen free radicals (.O2-, .OH, 1O2) and their by-products (H2O2, HOCl), generated by electrolysis of Krebs-Henseleit buffer, were determined in vitro by the DPD (N,N-diethyl-p-phenylenediamine) colorimetric method and ex vivo by quantifying cardiodynamic variables of the isolated perfused rat heart. Purified ceruloplasmin (1 mumol/l) displayed a high antioxidant capacity in vitro (89.2%), while the scavenging capacity of superoxide dismutase (SOD) in equimolar concentrations was 38.1%. A relatively high scavenging activity (72.1%) was observed with bovine serum albumin (BSA). A control group of Langendorff isolated rat hearts (n = 8) was submitted to electrolysis (10 mA, for 1 min) without treatment, whereas the treated groups were perfused with ceruloplasmin, SOD or BSA (1 mumol/l) in the inflow cannula for 5 min before, during, and 5 min after electrolysis. The cardioprotective effect afforded by ceruloplasmin (83-89%) was higher than that observed with the same optimal dose of 1 mumol/l SOD (20-45%). With BSA, no protection was observed ex vivo. Particularities in scavenging specificities and mechanisms seem to explain the important differences between in vitro and ex vivo antioxidant capacities for these proteins.

Antiarrhythmic effects of ceruloplasmin during reperfusion in the ischemic isolated rat heart.

The ability of ceruloplasmin, an important serum antioxidant, to reduce the vulnerability of the isolated rat heart to reperfusion arrhythmias has been investigated. Bovine plasma ceruloplasmin was purified by chromatography on aminoethyl-agarose. Isolated rat hearts were submitted to 15 min of regional ischemia and 10 min of reperfusion. The dose-effect relationship and the role of ceruloplasmin conformational integrity in cardioprotection were established by treatment of ischemic hearts with ceruloplasmin at various concentrations (0.25, 0.5, 1, and 2 microM) and at different degrees of conformational integrity (A610/A280 = 0.02, 0.04, and 0.06), 5 min before reperfusion. Deferoxamine (20-500 microM) was used as a positive control. As negative controls we used chemically inactivated ceruloplasmin (1 microM), heat-denatured ceruloplasmin (1 microM), and albumin (1-4 microM). In the control group during the first 5 min of reperfusion, the incidence of total ventricular fibrillation was 100% and of irreversible ventricular fibrillation was 83%. The incidence of reversible and irreversible ventricular fibrillation was significantly decreased in the ceruloplasmin-treated groups in both a dose and molecular integrity dependent manner. Ceruloplasmin had no effect on the incidence of ventricular tachycardia. Deferoxamine reduced the incidence of ventricular fibrillation to the same degree as ceruloplasmin but at concentrations much higher than those of ceruloplasmin. Chemically inactivated ceruloplasmin, heat-denatured ceruloplasmin, and albumin had no protective effects on reperfusion-induced arrhythmias.

Joint chromatographic purification of bovine serum ceruloplasmin and amineoxidase.

A purification procedure leading to a joint separation of two serum copper-enzymes: ceruloplasmin (EC 1.16.3.1) and amineoxidase (EC 1.4.3.6), is described. Both enzymes are obtained in electrophoretically homogeneous form and their specific activities are higher than those obtained by previously described purification techniques. Two common steps: precipitation of bovine plasma proteins with ammonium sulphate (at 35% and 55% saturation) followed by column chromatography on AE-Agarose (obtained by treatment of agarose beads with 1-chloro-2-ethylamine), lead to an electrophoretically homogeneous ceruloplasmin. At the same time, the ceruloplasmin-free protein preparation eluted in a first peak, following further Q-Sepharose and Con A-Sepharose chromatography, leads to purified bovine serum amine oxidase (BSAO) with an improved yield. The emphasis was given to a mutual improving effect as a consequence of the integration of the two enzymes purification procedures.