ramipril and apstatin

Bradykinin is a potent vasoactive peptide that is known to elicit a number of biological responses. A number of peptidases have been identified to possess kininase activity, the inhibition of which increases the availability and effectiveness of kinins. We wished to determine the cardioprotective actions of an aminopeptidase P inhibitor, apstatin alone and in combination with enalapril/lisinopril/ramipril in an in vivo rat model of acute ischemia (30 min) and reperfusion (4 h). Myocardial infarction was produced by occlusion of the left anterior descending coronary artery for 30 min followed by 4 h of reperfusion. Infarct size was measured by using the staining agent 2,3,5-triphenyl tetrazolium chloride (TTC). Lipid peroxide levels in serum and in heart tissue were estimated spectrophotometrically. A lead II electrocardiogram was monitored at various intervals throughout the experiment. Infarct size was reduced to a greater extent with apstatin and with combined inhibition it was further reduced. Infarct size reduction obtained with the combined inhibition came to normal with the prior administration of B2 bradykinin antagonist HOE140 suggests the involvement of bradykinin in the cardioprotective actions of apstatin.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Bradykinin Receptor Antagonists; Drug Therapy, Combination; Enalapril; Female; Heart Rate; Lipid Peroxides; Lisinopril; Male; Malondialdehyde; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peptides; Ramipril; Rats; Rats, Sprague-Dawley; Time Factors

1. Inhibitors of the angiotensin converting enzyme (ACE) have been shown to exert their cardioprotective actions through a kinin-dependent mechanism. ACE is not the only kinin degrading enzyme in the rat heart. 2. Since aminopeptidase P (APP) has been shown to participate in myocardial kinin metabolism to the same extent as ACE, the aims of the present study were to investigate whether (a) inhibition of APP leads to a reduction of myocardial infarct size in a rat model of acute ischaemia and reperfusion, (b) reduction of infarct size is mediated by bradykinin, and (c) a combination of APP and ACE inhibition leads to a more pronounced effect than APP inhibition alone. 3. Pentobarbital-anaesthetized rats were subjected to 30 min left coronary artery occlusion followed by 3 h reperfusion. The APP inhibitor apstatin, the ACE-inhibitor ramiprilat, or their combination were administered 5 min before ischaemia. Rats receiving HOE140, a specific B(2) receptor antagonist, were pretreated 5 min prior to enzyme inhibitors. Myocardial infarct size (IS) was determined by tetrazolium staining and expressed as percentage of the area at risk (AAR). 4. IS/AAR% was significantly reduced in rats that received apstatin (18+/-2%), ramiprilat (18+/-3%), or apstatin plus ramiprilat (20+/-4%) as compared with those receiving saline (40+/-2%), HOE (43+/-3%) or apstatin plus HOE140 (49+/-4%). 5. Apstatin reduces IS in an in vivo model of acute myocardial ischaemia and reperfusion to the same extent than ramiprilat. Cardioprotection achieved by this selective inhibitor of APP is mediated by bradykinin. Combined inhibition of APP and ACE did not result in a more pronounced reduction of IS than APP-inhibition alone.

Topics: Aminopeptidases; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Bradykinin; Bradykinin Receptor Antagonists; Heart; Heart Rate; Hemodynamics; Kinins; Male; Myocardial Infarction; Myocardium; Peptides; Protease Inhibitors; Ramipril; Rats; Rats, Wistar; Receptor, Bradykinin B2

Aminopeptidase P and angiotensin-converting enzyme (ACE) are responsible for the metabolism of exogenously administered bradykinin in the coronary circulation of the rat. It has been shown that ACE inhibitors decrease cytosolic enzyme release from the ischemic rat heart and reduce reperfusion-induced ventricular arrhythmias by increasing endogenous levels of bradykinin. It was hypothesized that the aminopeptidase P inhibitor apstatin could do the same. In an isolated perfused rat heart preparation subjected to global ischemia and reperfusion, both apstatin and ramiprilat (an ACE inhibitor) significantly decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release. The difference between the postischemia and preischemia levels of released CK was reduced 68% by apstatin and 68% by ramiprilat compared with control. The corresponding reductions in LDH release were 74% for apstatin and 81% for ramiprilat. A combination of the inhibitors was not significantly better than either one alone. Apstatin and ramiprilat also significantly reduced the duration of reperfusion-induced ventricular fibrillation by 69 and 61%, respectively. The antiarrhythmic effect of apstatin was reversed by HOE140, a bradykinin B2-receptor antagonist, suggesting that apstatin is acting by potentiating endogenously formed bradykinin. The results demonstrate that the aminopeptidase P inhibitor apstatin is cardioprotective in this model of cardiac ischemia/ reperfusion injury.

Topics: Adrenergic beta-Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Arrhythmias, Cardiac; Bradykinin; Cardiovascular Agents; Creatine Kinase; Drug Interactions; In Vitro Techniques; L-Lactate Dehydrogenase; Male; Peptides; Perfusion; Protease Inhibitors; Ramipril; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ventricular Fibrillation

Bradykinin (Bk), which is produced locally in the heart, exhibits potent cardioprotective effects. However, these effects appear to be limited by rapid degradation of the peptide. To determine the mechanism of Bk metabolism in the coronary circulation, [3H]Bk was perfused through the isolated rat heart via the aorta in the presence and absence of specific peptidase inhibitors. The radiolabeled metabolites were collected from the pulmonary artery and then separated, identified, and quantified by reversed-phase high-performance liquid chromatography (HPLC) by using a radioactive flow detector. In the absence of inhibitors, only 45 +/- 2% of the radioactivity eluted from the coronary circulation as intact [3H]Bk. The chromatograms suggested that Bk was being hydrolyzed at the Arg1-Pro2 bond by aminopeptidase P and at the Pro7-Phe8 bond by angiotensin-converting enzyme. When the aminopeptidase P inhibitor, apstatin (200 microM), was coperfused with [3H]Bk, cleavage at the Arg1-Pro2 bond was blocked and the amount of intact [3H]Bk in the perfusate increased to 57 +/- 5% (p < 0.05 vs. control). Coperfusion with the angiotensin-converting enzyme inhibitor, ramiprilat (0.5 microM), alone blocked cleavage at the Pro7-Phe8 bond and increased intact [3H]Bk to 75 +/- 3% (p < 0.001 vs. control). When both apstatin and ramiprilat were present, almost all of the radioactivity (96 +/- 1%) eluted as intact [3H]Bk (p < 0.01 vs. ramiprilat alone). The results indicate that the degradation of Bk in the rat coronary circulation can be fully accounted for by aminopeptidase P (approximately 30%) and angiotensin-converting enzyme (approximately 70%).

Topics: Aminopeptidases; Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Coronary Circulation; Coronary Vessels; Hydrolysis; In Vitro Techniques; Male; Peptides; Peptidyl-Dipeptidase A; Perfusion; Protease Inhibitors; Ramipril; Rats; Rats, Sprague-Dawley

1. Bradykinin (BK) has been shown to exert cardioprotective effects which are potentiated by inhibitors of angiotensin I-converting enzyme (ACE). In order to clarify the significance of ACE within the whole spectrum of myocardial kininases we investigated BK degradation in the isolated rat heart. 2. Tritiated BK (3H-BK) or unlabelled BK was either repeatedly perfused through the heart, or applied as an intracoronary bolus allowing determination of its elution kinetics. BK metabolites were analysed by HPLC. Kininases were identified by ramiprilat, phosphoramidon, diprotin A and 2-mercaptoethanol or apstatin as specific inhibitors of ACE, neutral endopeptidase 24.11 (NEP), dipeptidylaminopeptidase IV and aminopeptidase P (APP), respectively. 3. In sequential perfusion passages, 3H-BK concentrations in the perfusate decreased by 39% during each passage. Ramiprilat reduced the rate of 3H-BK breakdown by 54% and nearly abolished [1-5]-BK generation. The ramiprilat-resistant kininase activity was for the most part inhibited by the selective APP inhibitor apstatin (IC50 0.9 microM). BK cleavage by APP yielded the intermediate product [2-9]-BK, which was rapidly metabolized to [4-9]-BK by dipeptidylaminopeptidase IV. 4. After bolus injection of 3H-BK, 10% of the applied radioactivity were protractedly eluted, indicating the distribution of this fraction into the myocardial interstitium. In samples of such interstitial perfusate fractions, 3H-BK was extensively (by 92%) degraded, essentially by ACE and APP. The ramiprilat- and mercaptoethanol-resistant fraction of interstitial kininase activity amounted to 14%, about half of which could be attributed to NEP. Only the product of NEP, [1-7]-BK, was continuously generated during the presence of 3H-BK in the interstitium. 5. ACE and APP are located at the endothelium and represent the predominant kininases of rat myocardium. Both enzymes form a metabolic barrier for the extravasated fraction of BK. Thus, only interstitial, but not intravascular concentrations of BK are increased by kininase inhibitors to the extent that a significant potentiation of BK effects could be explained. NEP contributes less than 5% to the total kininase activity, but is the only enzyme which is exclusively present in the interstitial space.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; In Vitro Techniques; Male; Myocardium; Peptides; Protease Inhibitors; Ramipril; Rats; Rats, Wistar; Tissue Distribution; Tritium