enalaprilat-anhydrous and retrothiorphan

To study the metabolism of bradykinin (BK) after a single passage through the coronary bed in isolated Langendorff rat hearts.. 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.. 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.. 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.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Chromatography, High Pressure Liquid; Coronary Circulation; Coronary Vessels; Enalaprilat; Hemodynamics; Immunoenzyme Techniques; In Vitro Techniques; Male; Neprilysin; Perfusion; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Thiorphan; Time Factors

The role of angiotensin-converting enzyme (ACE) in the metabolism of bradykinin (BK) has been studied in several tissues. However, and contrary to angiotensin I, the metabolism of BK at the cardiac level has not been investigated. In this study, we define the participation of ACE in the carboxy-terminal degradation of BK in heart membranes of the dog, human, rabbit, and rat. The calculation of the kinetic parameters characterizing the metabolism of BK and the generated des-Arg9-BK can be summarized as follows: the half-life (t1/2) of BK [dog (218 +/- 32 s) > human (143 +/- 9 s) = rat (150 +/- 4 s) > rabbit (22 +/- 2 s)] and of des-Arg9-BK [dog (1,042 +/- 40 s) > human (891 +/- 87 s) > rat (621 +/- 65 s) > rabbit (89 +/- 8 s)] both showed significant differences according to species. Enalaprilat, an ACE inhibitor, significantly prevented the rapid degradation of BK and des-Arg9-BK in all species studied, whereas retrothiorphan, a neutral endopeptidase inhibitor, and losartan, an angiotensin II type I receptor antagonist, did not affect this metabolism. The relative importance of ACE in the cardiac metabolism of BK was species related: dog (68.4 +/- 3.2%) = human (72.2 +/- 2.0%) > rabbit (47.7 +/- 5.0%) = rat (45.3 +/- 3.9%). ACE participation in the metabolism of des-Arg9-BK was as follows: rabbit (57.0 +/- 4.0%) > dog (39.9 +/- 8.8%) = human (25.4 +/- 5.5%) = rat (36.0 +/- 7.0%). The participation of cardiac kininase I (carboxypeptidase M) in the transformation of BK into des-Arg9-BK was minor: human (2.6 +/- 0.1%) > dog (0.9 +/- 0.1%) = rabbit (1.0 +/- 0.1%) = rat (1.0 +/- 0.1%). These results demonstrate that ACE is the major BK-degrading enzyme in cardiac membranes. However, the metabolism of exogenous BK by heart membranes is species dependent. Our observations could explain some discrepancies regarding the contribution of kinins in the cardioprotective effects of ACE inhibitors.

Topics: Amino Acid Sequence; Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies; Bradykinin; Cross Reactions; Dogs; Enalaprilat; Humans; Kinetics; Losartan; Myocardium; Peptidyl-Dipeptidase A; Protease Inhibitors; Rabbits; Rats; Species Specificity; Substrate Specificity; Thiorphan

These experiments compare the effects of a neutral endopeptidase inhibitor, retrothiorphan, 1-[(1-mercaptomethyl-2-phenyl)ethyl]amino-1-oxopropanoic acid, a converting enzyme inhibitor, enalaprilat, and the combination of the two inhibitors on changes in blood pressure and renal function induced by exogenous and endogenous bradykinin in deoxycorticosterone acetate (DOCA)-salt rats. Enalaprilat potentiated the exogenous bradykinin-induced hypotensive responses while retrothiorphan potentiated the effects on urinary cyclic-GMP (cGMP) and bradykinin. The combination potentiated the exogenous bradykinin-induced hypotensive effects and the bradykinin-induced urinary excretion of cGMP, bradykinin and prostaglandin. The bradykinin B2 receptor antagonist, Hoe 140, had no effect on the enalaprilat- and retrothiorphan-induced changes in blood pressure and renal function. In conclusion, while angiotensin-converting enzyme and neutral endopeptidase are involved in the vascular and renal catabolism of exogenous bradykinin, the effects of the peptidase inhibitors do not appear to depend on the protection of endogenous bradykinin under acute conditions in DOCA-salt rats.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Bradykinin; Cyclic GMP; Enalaprilat; Hypertension; Kidney; Male; Neprilysin; Rats; Rats, Wistar; Thiorphan

Neutral endopeptidase inhibitors (NEPI) potentiate the hypotensive effect of converting enzyme inhibitors (CEI) in conscious spontaneously hypertensive rats (SHR) but the mechanism of this potentiation remains unknown. The present study assesses the hemodynamic effects of a CEI (enalaprilat 1 mg/kg; n = 9), a NEPI (retrothiorphan 25 mg/kg + 25 mg/kg/h; n = 9) and the combination (CEI+NEPI; n = 9) versus a control group (n = 9) in anesthetized spontaneously hypertensive rats. CEI alone induced a significant hypotensive effect due to a decrease (-35.1%) in total peripheral resistance (TPR), with no significant increase in cardiac output (CO). NEPI alone had a slight hypotensive effect due to a small decrease in CO. CEI+NEPI decreased the mean arterial pressure to the same extent (-26.7%) as the CEI-induced hypotensive effect, decreased TPR (-44.4%) and induced an increase in CO (+38.2%) with an increase in heart rate. In summary, NEPI combined with CEI induces large decreases in blood pressure and in TPR which do not significantly differ from the CEI-induced effects. It also induces increases in heart rate and in cardiac output in anesthetized SHR.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Drug Therapy, Combination; Enalaprilat; Heart Rate; Hemodynamics; Hypertension; Kinetics; Neprilysin; Rats; Rats, Inbred SHR; Thiorphan; Time Factors

Angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) are implicated in the metabolism of several peptides involved in blood pressure and sodium homeostasis control, such as angiotensins, atrial natriuretic factor (ANF), bradykinin and endothelin. The effects of a highly selective NEP inhibitor (NEPI), retrothiorphan, of a converting enzyme inhibitor (CEI), enalaprilat, and of the combination, CEI + NEPI, were assessed in deoxycorticosterone acetate (DOCA)-salt hypertensive rats, spontaneously hypertensive rats (SHRs) and renovascular hypertensive rats. NEPI increased diuresis, natriuresis and urinary cyclic GMP (cGMP), ANF and bradykinin in the three models. NEPI decreased blood pressure in DOCA-salt hypertensive rats only, whereas CEI decreased blood pressure in SHRs and renovascular hypertensive rats only and increased plasma renin. CEI had no effect on urinary aldosterone or bradykinin in any of the three models. CEI + NEPI increased diuresis and natriuresis in DOCA-salt hypertensive rats and SHRs, and increased urinary cGMP, ANF and bradykinin and plasma renin levels. CEI and NEPI interacted significantly to decrease blood pressure and to increase urinary cGMP in SHRs only. Hence, NEPI increases diuresis, natriuresis and urinary cGMP, ANF and bradykinin in experimental hypertension, whereas CEI acts on blood pressure and increases in plasma renin in SHRs and renovascular hypertensive rats. The significant interaction between CEI and NEPI to decrease blood pressure in SHRs indicates that simultaneous blockade of the two metallopeptidases results in potentiation of the hypotensive effect and that the SHRs appear to be a good model for studying NEP and ACE coinhibition. Finally, NEP rather than ACE appears to be involved in bradykinin renal catabolism in experimental hypertension.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Desoxycorticosterone; Diuresis; Enalaprilat; Hypertension, Renovascular; Kidney; Male; Natriuresis; Neprilysin; Protease Inhibitors; Rats; Rats, Inbred SHR; Rats, Wistar; Sodium Chloride; Sulfhydryl Compounds; Thiorphan