Measuring Angiotensin-I Converting Enzyme Inhibitory Activity by Micro Plate Assays: Comparison Using Marine Cryptides and Tentative Threshold Determinations with Captopril and Losartan
Yesmine Ben HendaAnis LabidiIngrid Fruitier‐ArnaudinNicolas BridiauRégis DelatoucheThierry MaugardJean-Marie PiotFredéric SannierValérie ThiéryStéphanie Bordenave-Juchereau
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To determine the angiotensin-I converting enzyme (ACE) inhibitory activity of marine cryptides, different methods were tested. ACE inhibition was measured using two synthetic substrates, (N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) and N-hippuryl-His-Leu hydrate salt (HHL)), and a natural one, angiotensin-I. The IC50 value (defined as the concentration of inhibitory molecule needed to inhibit 50% of the ACE activity) of the reference synthetic inhibitor captopril was in the nanomolar range (1.79-15.1 nM) when synthetic substrates were used, whereas it exhibited IC50 of micromolar range (16.71 μM) with angiotensin-I. We chose losartan, an antagonist of angiotensin-II receptor as negative control for the ACE inhibition. Losartan was also able to inhibit ACE whatever the substrate tested, with IC50 of micromolar range (17.13-146 μM). We defined this value as a limit above which molecules are not showing in vitro ACE inhibitory activity. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK), and Val-Ala-Pro (VAP) were tested as inhibitors of ACE with synthetic and natural substrates. IC50 displayed were substrate-dependent. With FAPGG as substrate, IW, VAP, KY, IY, AP, AKK, and VIY show IC50 values over the IC50 value of losartan and should not be considered as inhibitors of ACE. VY, VW, KW, and LKP exhibited IC50 value lower than the IC50 value of losartan for all substrates tested and were thus considered as good candidates for effectively decreasing hypertension. It appears that the comparison of IC50 is not consistent when IC50 values are obtained with different substrates and different methods. In vitro ACE inhibitory activity assays should always include various ACE substrates and references such as captopril and a negative control to obtain data reliable to discriminate ACE inhibitory peptides.Keywords:
Captopril
We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33% of the initial values, P<0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 µmol/100 g body weight as a bolus, iv, plus a 30-min infusion of 0.018 µmol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P<0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 ± 0.28 mM, angiotensin II, N = 9 vs 6.4 ± 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.
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Delayed recovery of hypertension after single dose losartan in angiotensin II-infused conscious rats
Objective In a conscious unrestrained rat model, it takes approximately 1 week for angiotensin II to increase blood pressure to maximum levels. We investigated the time required for hypertension to fully recover after acute angiotensin II receptor blockade in this angiotensin II dependent hypertensive model. Design Conscious unrestrained rats (n = 8) infused with 10 ng/kg per min angiotensin II for 21 days received losartan (10 mg/kg) on day 17 of angiotensin II infusion. Mean arterial pressure (MAP) and heart rate were monitored continuously. The acute pressor response to 50 ng/kg per min angiotensin II was monitored for 2 h on days 15, 17, 18, 19 and 20 of angiotensin II infusion. Plasma renin concentration (PRC) was measured daily. Results Angiotensin II increased MAP acutely by 26 ± 2 mmHg and by a further 23 ± 4 mmHg between days 4 and 8. Losartan acutely reduced MAP by 75 ± 2 mmHg; 24 h later MAP had partially recovered but remained suppressed by 47 ± 3 mmHg. MAP had not fully recovered 4 days later. Some 2 h after losartan, the acute pressor response to angiotensin II had fallen from 24 ± 2 mmHg to zero. This recovered to 13 ± 5 and 28 ± 2 mmHg 24 and 48 h post losartan. After losartan PRC rose from 0.1 ± 0.05 to above 1 ng/ml per h for less than 24 h. Conclusion A single dose of losartan reverses both the fast and slow pressor effects of continuous angiotensin II infusions. While losartan is metabolized, the fast vasoconstrictor effect recovers quickly but the slow pressor effect takes almost a week to build up again to maximum levels. Since the slow pressor effect is mediated via the AT1 receptor, any means of blocking the renin–angiotensin system is likely to keep blood pressure below maximum hypertensive levels for several days after the drug has disappeared from the circulation.
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OBJECTIVE To observe the effects of losartan and c aptopril on renal structure and function in spontaneously hypertensive rats(SHR) .METHODS Thirty SHRs were gorged with losartan (30 mg·kg -1 ·d -1 ), captopril (100 mg·kg -1 ·d -1 ),losartan (15 mg·k g -1 ·d -1 ) + captopril (50 mg·kg -1 ·d -1 ) or water respec tively for 15 weeks in the treating and control groups.The blood pressure and pr oteinuria were assessed, the levels of angiotensin Ⅱ(AngⅡ) and transforming gr owth factor(TGF)β 1 were detected, and renal tissues were examined routinely b y optical and electron microscopy respectively.RESULTS The e xcretion of proteinuria was significantly decreased in the treating groups compa red with the control group.There was a decrease TGFβ 1 and collagen Ⅳ deposit ion in the treatment group, and further lower in losartan + captopril group.Losa rtan and captopril attenuated the injuries of small arteries.CONCLUSI ON The combination of losartan with captopril may attenuate renal damag e,they had synergistic action when used together.
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SUMMARY 1. The effects of exogenous angiotensins II and III (50 pmol/min i.v.) on plasma renin release following captopril injection (5 mg/kg, i.v.) were studied in anaesthetized Sprague‐Dawley rats, to determine whether angiotensin II blockade is the major mechanism by which captopril induces renin release. 2. Captopril produced a 12‐fold increase in plasma renin concentration compared with saline‐injected controls. This was completely reversed by pre‐infusion of angiotensin II or III. 3. The fall in blood pressure following captopril treatment was also abolished by angiotensins II and III pre‐infusion. Noradrenaline pre‐infusion (200–800 ng/min, i.v.) also prevented the captopril‐induced hypotension but did not alter the rise in plasma renin. 4. Ureteric ligation did not significantly reduce captopril‐induced renin release suggesting that acute changes in sodium excretion or delivery of electrolyte to the macula densa were not involved in renin release. 5. These findings suggest that captopril induces renin release by inhibiting angiotensin II feedback control of renin secretion and that angiotensin III may also modulate renin release.
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This study investigated the ability of the selective angiotensin II (AII)-type (AT)1 receptor antagonist losartan to reverse the fast (< 30 sec) pressor effect of AII, and the hypertension produced by chronic (2 weeks) i.v. infusion of AII (AII hypertension). We hypothesized the following: if AII hypertension is caused solely by stimulation of AT1 receptors mediating the fast pressor effect, then the time course of the antihypertensive effect of losartan in AII hypertension should parallel the time course of losartan inhibition of pressor responses to acute, bolus injection of AII. Thus, in one group of rats, pressor responses to bolus injections of AII (10 ng, n = 10) were measured before and subsequently at numerous time points after losartan administration (3 mg.kg-1 i.v.). Other groups of rats received continuous infusions of AII i.v. for 15 days at 2 ng.min-1 (n = 8), 4 ng.min-1 (n = 8) or 10 ng.min-1 (n = 6). On days 2, 7 and 12 of the AII infusion, rats received a bolus injection of losartan (3 mg.kg-1, i.a.). Mean arterial pressure (MAP) was then measured at numerous time points after losartan administration. Within 5 min of administration, losartan caused almost complete inhibition of fast pressor responses to acute injections of AII, and the magnitude of this inhibition did not change for over 24 hr. On the other hand, in AII hypertension, losartan lowered MAP significantly within 5 min only in rats receiving 10 ng.min-1 AII, but in all three groups caused a slower decline in MAP that peaked around 2 hr after losartan injection.(ABSTRACT TRUNCATED AT 250 WORDS)
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Losartan (DuP 753) and PD123177 are nonpeptide angiotensin (ANG) receptor ligands for subtypes of ANG II receptors ANG II-1 and ANG II-2, respectively. We examined the effects of losartan and PD123177 on dose – mean arterial pressure (MAP) response curves for ANG II and ANG III in eight groups (n = 6 each) of conscious rats. Saline (0.9% NaCl), losartan (1 × 10 −6 and 9 × 10 −6 mol/kg), and PD123177 (2 × 10 −5 mol/kg) were i.v. bolus injected 15 min before the construction of ANG II dose–response curves in groups I, II, III, and IV, respectively. Groups V–VIII were treated similarly to I–IV except that ANG III was given in place of ANG II. Losartan dose dependently shifted the dose–response curves of ANG II and ANG III to the right with similar dissociation constants (−log K I of 6.6 ± 0.7 and 6.6 ± 0.1 mol/kg, respectively) and no change in the maxima. PD123177 affected neither maximum MAP nor ED 50 values for ANG II or ANG III. Our results show that losartan but not PD123177 is a competitive antagonist of the MAP effects of ANG II and ANG III.Key words: nonpeptide angiotensin receptor antagonist, angiotensin II, angiotensin III, blood pressure, losartan.
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OPTIMAAL (Optimal Trial in Myocardial Infarction with the Angiotensin II Antagonist Losartan) is the first major study to compare an angiotensin II Type 1 antagonist losartan (Cozaar™, Merck) with an ACE inhibitor captonpril (Capoten™, Elan) after myocardial infarction in patients with left ventricular dysfunction. Patients were assigned to a target dose of losartan 50 mg/day and captopril 50 mg t.i.d., as tolerated. The primary end point was all-cause mortality and there were 499 (18%) and 447 (16%) deaths in the losartan and captopril group, respectively (p = 0.07). However, there were significantly more cardiovascular deaths with losartan (420, 15%) than with captopril (363, 13%; p = 0.03). Losartan was better tolerated than captopril with fewer patients discontinuing medication (17 versus 23% for losartan and captopril, respectively). In conclusion, if tolerated, captopril should remain the preferred treatment for patients after complicated acute myocardial infarction.
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OPTIMAAL (Optimal Trial in Myocardial Infarction with the Angiotensin II Antagonist Losartan) is the first major study to compare an angiotensin II Type 1 antagonist losartan (Cozaar™, Merck) with an ACE inhibitor captonpril (Capoten™, Elan) after myocardial infarction in patients with left ventricular dysfunction. Patients were assigned to a target dose of losartan 50 mg/day and captopril 50 mg t.i.d., as tolerated. The primary end point was all-cause mortality and there were 499 (18%) and 447 (16%) deaths in the losartan and captopril group, respectively (p = 0.07). However, there were significantly more cardiovascular deaths with losartan (420, 15%) than with captopril (363, 13%; p = 0.03). Losartan was better tolerated than captopril with fewer patients discontinuing medication (17 versus 23% for losartan and captopril, respectively). In conclusion, if tolerated, captopril should remain the preferred treatment for patients after complicated acute myocardial infarction.
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