Specific Angiotensin-converting Enzyme Inhibitor Research Articles

Selective Inhibition of the C-Domain of ACE (Angiotensin-Converting Enzyme) Combined With Inhibition of NEP (Neprilysin): A Potential New Therapy for Hypertension.

[Figure: see text].

Purification and characterization of angiotensin-converting enzyme (ACE) from sheep lung.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) in the renin-angiotensin system regulates blood pressure by catalyzing angiotensin I to the vasoconstrictor angiotensin II. In this study, the ACE was purified and characterized from sheep lung. The kinetic properties of the ACE were designated. The inhibition effect of captopril, a specific ACE inhibitor, was determined. ACE was purified from sheep lung using the affinity chromatography method in one step. NHS-activated Sepharose 4 Fast Flow as column filler and lisinopril as a ligand in this method used. The molecular weight and purity of ACE were designated using the SDS-PAGE method. Optimum temperature and optimum pH were found for purified ACE. KM and Vmax values from Lineweaver–Burk charts determined. The inhibition type, IC50, and Ki values of captopril on purified ACE were identified. ACE was 6405-fold purified from sheep lung by affinity chromatography in one step and specific activity was 16871 EU/mg protein. The purity and molecular weight of ACE were found with SDS-PAGE and observed two bands at around 60 kDa and 70 kDa on the gel. Optimum temperature and optimum pH were designated for purified ACE. Optimum temperature and pH were found as 40 °C and pH 7.4, respectively. Vmax and KM values were calculated to be 35.59 (µmol/min).mL−1 and 0.18 mM, respectively. IC50 value of captopril was found as 0.51 nM. The inhibition type of captopril was determined as non-competitive from the Lineweaver–Burk graph and the Ki value was 0.39 nM. As a result, it was observed in this study that the ACE enzyme can be successfully purified from sheep lungs in one step. Also, it was determined that captopril, which is a specific ACE inhibitor, has a significant inhibitory effect with a very low IC50 value of 0.51 nM.

A new RP\u2013HPLC method for simultaneous quantification of perindopril erbumine, indapamide, and amlodipine besylate in bulk and pharmaceutical dosage form

BackgroundPerindopril erbumine is a specific inhibitor of angiotensin-converting enzyme, indapamide is the one providing thiazide diuretic effect, and amlodipine besylate is a calcium antagonist which belongs to the dihydropyridines which helps to maintain the pressure of the blood in the patient having arterial hypertension. The literature survey discloses that only one method is available for the estimation of the combination in the quantitative analytical liquid chromatographic method. Moreover to this, the literature review also reveals that HPTLC, UV spectroscopy, and HPLC methods are available for the analysis of either of the two in combination. Hence, our area of interest is to develop and validate the RP-HPLC in order to quantify perindopril erbumine, indapamide, and amlodipine besylate simultaneously in bulk and formulation.ResultSensitive and accurate RP-HPLC method was developed for the simultaneous estimation of indapamide, perindopril erbumine, and amlodipine besylate in bulk and available as triplixam-marketed tablet dosage form which is a combination of these drugs. The Phenomenex C-18 column (250 mm × 4.6 mm, 5 μm) was used as a stationary phase, and acetonitrile: methanol: water (30:20:50, v/v/v) was found to be optimized mobile phase which was further adjusted to pH 3.0 by utilizing 1.0% orthophosphoric acid; the flow rate kept was 1 ml/min and experiments were performed using PDA detector. The common detection wavelength for all the three APIs was found to be 215.0 nm. The method was validated as per ICH Q2 (R1). The linearity range for amlodipine besylate was found to be 0.500–9.500 μg/ml; for perindopril erbumine was found to be 0.400–7.600 μg/ml, and for indapamide was found to be 0.125–2.375 μg/ml. The correlation coefficient was found to be more than 0.9975 for all three of them, whereas the mean percentage recovery was found to be 99.52–100.71%, 99.49–100.89%, and 99.90–100.78%, respectively.ConclusionThe proposed RP-HPLC method is found to be accurate and robust enough to estimate the perindopril erbumine, indapamide, and amlodipine besylate simultaneously in bulk and available tablet dosage form of combination.

Primacy of Chymase over Angiotensin Converting Enzyme in the Production of Angiotensin II in Rat Bone Marrow Tissue

BackgroundThe bone marrow is attracting attention as not only the nidus of circulating blood cells but the source of stem and progenitor cell‐based therapy. Renin‐angiotensin system (RAS) and angiotensin II (Ang II) receptor type 1 in bone marrow participate in hematopoiesis, a target in the evolution of inflammatory processes involved in cardiovascular disease pathogenesis. Extending the original demonstration of RAS components in the rat bone marrow (Strawn et al. Br J Haematol. 2004;126:120–6), this study investigated the existence of an Ang II‐forming pathway in which chymase rather than angiotensin‐converting enzyme (ACE) cleaves the peptide from the newly described substrate angiotensin‐(1‐12) [Ang‐(1‐12)] which functions as an endogenous tissue substrate for the direct formation of Ang II by chymase.MethodsBone marrow tissue was obtained from the tibia of twelve weeks‐old Sprague Dawley rats following systemic perfusion with phosphate buffered saline with or without the secondary addition of Zamboni's fixative. Bone marrow tissues, homogenized with 0.05 M Tris‐HCl and centrifuged at 28,000 g, were subjected to immunoblot and chymase enzymatic activity using high‐performance liquid chromatography (HPLC). Additional samples were prepared for quantitative RT‐PCR analysis normalized to GAPDH. All data were analyzed using GraphPad PRISM 7.0 with P<0.05 considered statistically significant.ResultsFluorescent immunohistochemistry showed the existence of immunoreactive (ir‐) Ang‐(1‐12) and ir‐chymase‐positive cells in rat's bone marrow. Immunoblots showed chymase protein expression in cell pellets but not in supernatants. Assessment of 125I‐Ang‐(1‐12) or 125I‐Ang I hydrolysis in cell pellets in the presence and absence of specific chymase or ACE inhibitors, revealed that production of Ang II by chymase was two‐thousand fold higher compared to ACE (4,531 ± 137 fmol/mg/min and 2.3 ± 0.3 fmol/mg/min, respectively, P<0.0001, n = 6). ANOVA and post‐hoc Tukey's test showed that gene expression of rat mast cell protease‐5 (rMCP‐5) was at least twice higher than rMCP‐1, rMCP‐2 and rMCP‐4.ConclusionThese studies reveal a major role of Ang‐(1‐12) and chymase as the critical pathway for Ang II‐formation in rat's bone marrow. The pleotropic actions of tissue chymase in the degradation of matricellular proteins, activation of matrix metalloproteinases, transforming growth factor‐β, and production of inflammatory cytokines posits this alternate pathway for Ang II‐production as most relevant in understanding and treating cardiovascular disease.Support or Funding InformationThis study was supported by the National Heart, Blood, Lung Institute of the NIH through HL‐051952 grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The angiotensin converting enzyme (ACE) inhibitor, captopril disrupts the motility activation of sperm from the silkworm, Bombyx mori

Angiotensin I-converting enzyme (also known as peptidyl dicarboxypeptidase A, ACE, and EC 3.4.15.1), which is found in a wide range of organisms, cleaves C-terminal dipeptides from relatively short oligopeptides. Mammalian ACE plays an important role in the regulation of blood pressure. However, the precise physiological functions of insect ACE homologs have not been understood. As part of our effort to elucidate new physiological roles of insect ACE, we herein report a soluble ACE protein in male reproductive secretions from the silkmoth, Bombyx mori. Seminal vesicle sperm are quiescent in vitro, but vigorous motility is activated by treatment with either a glandula (g.) prostatica homogenate or trypsin in vitro. When seminal vesicle sperm were pre-incubated with captopril, a strong and specific inhibitor of mammalian ACE, and then stimulated to initiate motility by the addition of the g. prostatica homogenate or trypsin, the overall level of acquired motility was reduced in an inhibitor-concentration-dependent manner. In the course of this project, we detected ACE-related carboxypeptidase activity that was inhibited by captopril in both the vesicular (v.) seminalis of the noncopulative male reproductive tract and in the spermatophore that forms in the female bursa copulatrix at the time of mating, just as in an earlier report on the tomato moth, Lacanobia oleracea, which belongs to a different lepidopteran species (Ekbote et al., 2003a). Two distinct genes encoding ACE-like proteins were identified by analysis of B. mori cDNA, and were named BmAcer and BmAcer2, respectively [the former was previously reported by Quan et al. (2001) and the latter was first isolated in this paper]. RT-qPCR and Western blot analyses indicated that the BmAcer2 was predominantly produced in v. seminalis and transferred to the spermatophore during copulation, while the BmAcer was not detected in the adult male reproductive organs. A recombinant protein of BmAcer2 (devoid of a signal peptide) that was expressed in Escherichia coli cells exhibited captopril-sensitive carboxypeptidase activities. Our findings show that the BmAcre2 gene encodes a secreted ACE protein included in the Bombyx seminal plasma. In particular, the silkworm ACE protein in the seminal fluid might be involved in the signaling pathway that leads to the activation and regulation of sperm motility.

Unspecified intestinal malabsorption in patients treated with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers: a retrospective analysis in primary care settings

ABSTRACTObjective: To investigate the possible relationship between the use of Angiotensin II Receptor Blockers (ARBs) or Angiotensin-Converting-Enzyme Inhibitors (ACE-i) and unspecified intestinal malabsorption (IM) within the Italian and German real-life context.Research design and methods: a retrospective cohort of patients with a new unspecified IM diagnosis during the period 1 January 2010–31 December 2013 was extracted from Italian IMS Health Longitudinal Patient Database and German IMS Disease Analyzer. Only patients with at least one prescription of ARB or ACE-i medication during the 6 months preceding the IM diagnosis were included and then followed up for 12 months to assess treatment exposure.Results: After stratification by year and molecule, the proportion of patients experiencing an unspecified IM diagnosis on total patients receiving ARBs or ACE-i ranged from 0% to 0.14%, showing no relevant differences between molecules and no time trends.Conclusions: this study indicates that ACE-i or ARBs were rarely associated to an unspecified IM diagnosis. No relevant difference between each specific ACE-i and ARB was highlighted.

The incidence of all-cause, cardiovascular and respiratory disease admission among 20,252 users of lisinopril vs. perindopril: A cohort study

BackgroundMajor international guidelines do not offer explicit recommendations on any specific angiotensin-converting enzyme inhibitor (ACEI) agent over another within the same drug group. This study compared the effectiveness of lisinopril vs. perindopril in reducing the incidence of hospital admission due to all-cause, cardiovascular disease and respiratory disease. MethodsAdult patients who received new prescriptions of lisinopril or perindopril from 2001 to 2005 in all public hospitals and clinics in Hong Kong were included, and followed up for ≥2years. The incidence of admissions due to all-cause, cardiovascular disease and respiratory disease were evaluated, respectively, by using Cox proportional hazard regression models. The regression models were constructed with propensity score matching to minimize indication biases. ResultsA total of 20,252 eligible patients with an average age of 64.5years (standard deviation 15.0) were included. The admission rate at 24months within the date of index prescription due to any cause, cardiovascular disease and respiratory disease among lisinopril vs. perindopril users was 24.8% vs. 24.8%, 13.7% vs. 14.0% and 6.9% vs. 6.3%, respectively. Lisinopril users were significantly more likely to be admitted due to respiratory diseases (adjusted hazard ratios [AHR]=1.25, 95% CI 1.08 to 1.43, p=0.002 at 12months; AHR=1.17, 95% CI 1.04 to 1.31, p=0.009 at 24months) and all causes (AHR=1.12, 95% CI 1.05 to 1.19, p<0.001 at 24months) than perindopril users. ConclusionsThese findings support intra-class differences in the effectiveness of ACEIs, which could be considered by clinical guidelines when the preferred first-line antihypertensive drugs are recommended.

Ramipril Affects Hydrogen Sulfide Generation in Mouse Liver and Kidney

Hydrogen sulfide (H2S) is a modulator of various physiological and pathological processes in the cardiovascular and nervous system and plays an important role in the regulation of gastrointestinal tract, liver and kidney function. The effect of the pleiotropic action of the tissue specific angiotensin-converting enzyme inhibitor (ACEI), ramipril, exceeds renin-angiotensin aldosterone system (RAAS) blockade and involves different biological mechanisms. The aim of the study is to assess the influence of ramipril on H2S production in mouse liver and kidneys. Thirty mice (CBA) of both sexes were given intraperitoneal injections of ramipril solutions--0.125 mg (5 mg/kg--group D1) and 0.25 mg (10 mg/kg--group D2) for 5 consecutive days at the same time of the day (10:30 am). The control group received physiological saline in portions of the same volume--0.2 ml. The measurements of the tissue concentration of H2S were performed using the modified spectrophotometric method of Siegel. There was a significant rise in the tissue concentration of H2S [microg/g] in livers of group D1 (2.70 +/- 0.02 vs 2.81 +/- 0.06; P = 0.03) and group D2 (2.70 +/- 0.02 vs 2.98 +/- 0.03; P < 0.001) and a significant decrease of H2S kidney tissue concentration in group D1 (3.35 +/- 0.06 vs 3.15 +/- 0.07; P = 0.02) and in group D2 (3.35 +/- 0.06 vs 2.89 +/- 0.03; P < 0.001). Our results show that ACEI ramipril affects hydrogen sulfide generation in mouse liver and kidneys.

THE USE OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS AFTER MYOCARDIAL INFARCTION. WHAT DO EVIDENCE-BASED MEDICINE DATA SPEAK FOR?

The main trails proven angiotensin converting enzyme (ACE) inhibitor effects on the life prognosis after myocardial infarction (MI) are described. Different tactics of ACE inhibitors therapy in post MI patients are presented. Approaches to the choice of a specific ACE inhibitor are discussed.

Physician gender is associated with the quality of type 2 diabetes care

Patient gender influences the quality of medical care whilst the role of physician gender is not well established. To investigate the influence of physician gender on quality of care in patients with type 2 diabetes. Cross-sectional study in 51 053 outpatients (48.6% male), treated by 3096 office-based physicians (66.3% male; 74.0% general practitioners, 21.8% internists and 4.2% diabetologists). Outcome measures included processes of care, intermediate outcomes and medical management. Quality of care measures were based on current ADA guidelines. Hierarchical regression models were used to avoid case-mix bias and to correct for physician-level clustering. Adjusted odds ratios were calculated controlling for age, gender, disease duration and presence of atherosclerotic disease. The patients of female physicians were more often women, more obese, older and had more often atherosclerotic disease (34% in the total cohort). The patients of female physicians more often reached target values in glycaemic control (HbA1c < 6.5%; OR 1.14; 1.05-1.24, P = 0.002), blood lipoproteins (LDL-C < 100 mg dL(-1); OR 1.16; 1.06-1.27, P = 0.002), and blood pressure (systolic values < 130 mmHg; OR 1.11; 1.02-1.22, P = 0.018). They were more likely to receive antihypertensive drug therapy in general (OR 1.35; 1.24-1.46, P < 0.0001) and angiotensin converting enzyme (ACE) inhibitors in particular (OR 1.17; 1.09-1.25, P < 0.0001). The patients of female physicians less often performed glucose self-monitoring (OR 0.83; 0.76-0.91, P < 0.0001) and less often received oral hypoglycaemic agents (OR 0.88; 0.82-0.95, P = 0.001). Physician gender influences quality of care in patients with type 2 diabetes. Female physicians provide an overall better quality of care, especially in prognostically important risk management.

Tissue angiotensin-converting enzyme inhibitors: Are they more effective than serum angiotensin-converting enzyme inhibitors?

Since their discovery in the 1980s, angiotensin-converting enzyme (ACE) inhibitors have been shown to decrease angiotensin formation, prevent breakdown of bradykinin, and may also act on peptides of the renin-angiotensin system. They are effective in reducing the risk of heart failure, myocardial infarction, and death from cardiovascular causes in patients with left ventricular systolic dysfunction or heart failure, and have been shown to reduce atherosclerotic complications in patients who have vascular disease without heart failure. They may preserve endothelial function and counteract initiation and progression of atherosclerosis. Broadly, ACE inhibitors can be divided into tissue specific or serum ACE inhibitors. Tissue-specific ACE inhibitors as a group are not superior to serum ACE inhibitors in the treatment of coronary artery disease. Pending direct comparator clinical trials between a tissue ACE inhibitor and a plasma ACE inhibitor, both ramipril and perindopril can be recommended for secondary risk prevention, based on the evidence.

An updated clinical primer on large artery mechanics: implications of pulse waveform analysis and arterial tonometry

The use of pulse wave analysis with arterial tonometry has accelerated over the last year. Despite approval from the US Food and Drug Administration in 2001 on the use of generalized transfer function to generate the central (aortic) pressure wave from the radial waveform, this technique is still questioned. This review summarizes major findings on (a) value of arterial tonometry in determining indices of cardiovascular function, (b) use of these indices in outcome and drug studies, (c) relevance to major trials on blood pressure reduction. Pulse pressure has emerged as a better predictor of cardiac ischemic events than systolic, diastolic, and mean brachial pressure. Central systolic and pulse pressure and augmentation index have shown an even better relation with cardiovascular events and with outcomes. The claim by specific angiotensin-converting enzyme inhibitor and angiotensin receptor blocker drugs of their benefits "beyond blood pressure lowering" has been challenged on the basis of greater reduction in central and aortic pressure compared with brachial pressure measured by cuff sphygmomanometer, as shown by the pREterax in regression of Arterial Stiffness in a contrOlled double-bliNd study. Augmentation index is higher in hypertension, is inversely related to body height, and can be reduced by exercise. Augmentation index shows a linear relation with age up to 60 years. Regrettably, recent major trials such as the Comparison of Amlodipine versus Enalapril to Limit Occurrences of Thrombosis, Prevention of Events with Angiotensin Converting Enzyme Inhibition, and Valsartan Antihypertensive Long-term Use Evaluation studies have not included pulse wave analysis to distinguish the relative benefit of different drugs. Pulse wave analysis will assist in a better understanding of hypertension as well as in establishing the extent of cardiovascular disease and for monitoring therapy.

Angiotensin II and Epidermal Growth Factor Induce Cyclooxygenase-2 Expression in Intestinal Epithelial Cells through Small GTPases Using Distinct Signaling Pathways

Colorectal carcinogenesis is a multistep process involving genetic mutations and alterations in rigorously controlled signaling pathways and gene expression that control intestinal epithelial cell proliferation, differentiation, and apoptosis. Cyclooxygenase-2 (COX-2) is aberrantly expressed in premalignant adenomatous polyps and colorectal carcinomas and is associated with increased epithelial cell proliferation, decreased apoptosis, and increased cell invasiveness. Currently, knowledge of the regulation of expression of COX-2 by endogenous cell-surface receptors is inadequate. Recently, in a non-transformed rat intestinal epithelial cell line (IEC-18), we showed induction of cell proliferation and DNA synthesis by angiotensin II (Ang II) via the endogenous Ang II type 1 receptor (Chiu, T., Santiskulvong, C., and Rozengurt, E. (2003) Am. J. Physiol. 285, G1-G11). We report that Ang II potently stimulated expression of COX-2 mRNA and protein as an immediate-early gene response through the Ang II type 1 receptor, correlating with an increase in prostaglandin I2 production. Ang II induced Cdc42 activation and filopodial formation. COX-2 expression was induced by epidermal growth factor (EGF), which activated Rac with lamellipodial formation. Inhibition of small GTPases by Clostridium difficile toxin B blocked COX-2 expression by Ang II and EGF. Inhibition of ERK activation by U0126 or PD98059 significantly decreased EGF-dependent COX-2 expression, but did not affect Ang II-dependent COX-2 expression. Conversely, inhibition of p38MAPK by SB202190 or PD169316 inhibited COX-2 expression by Ang II, but did not block COX-2 induction by EGF. Ang II caused Ca2+ mobilization. Inhibition of Ca2+ signaling by 2-aminobiphenyl borate blocked Ang II-dependent COX-2 expression. EGF did not induce Ca2+ mobilization, and 2-aminobiphenyl borate did not inhibit EGF-dependent COX-2 expression. Inhibition of COX-2 expression correlated with inhibition of prostaglandin I2 production. Luciferase promoter assays showed that Ang II-dependent transcriptional activation of the COX-2 promoter was dependent on activation of small GTPases and p38(MAPK) and on Ca2+ signaling via the cAMP-responsive element/activating transcription factor cis-acting element.

Mortality rates in elderly patients who take different Angiotensin-Converting Enzyme inhibitors after acute myocardial infarction: A class effect?

Survival benefits in the first year after acute myocardial infarction in patients 65 years of age or older seem to differ according to the specific angiotensin-converting enzyme inhibitor prescribe...

Mortality Rates in Elderly Patients Who Take Different Angiotensin-Converting Enzyme Inhibitors after Acute Myocardial Infarction: A Class Effect?

Several randomized, controlled trials show that angiotensin-converting enzyme (ACE) inhibitors improve survival in patients who have had an acute myocardial infarction. However, existing data from trials do not address whether all ACE inhibitors benefit patients similarly. To evaluate whether all ACE inhibitors are associated with similar mortality in patients 65 years of age or older who have had an acute myocardial infarction. Retrospective cohort study that used linked hospital discharge and prescription databases containing information on 18 453 patients 65 years of age or older who were admitted for an acute myocardial infarction between 1 April 1996 and 31 March 2000. 109 hospitals in Quebec, Canada. 7512 patients who filled a prescription for an ACE inhibitor within 30 days of discharge and who continued to receive the same drug for at least 1 year. The association between the specific drugs and clinical outcomes was measured by using Cox proportional hazards models, with adjustment for demographic, clinical, physician, and hospital variables and dosage categories, represented by time-dependent variables. Enalapril, fosinopril, captopril, quinapril, and lisinopril were associated with higher mortality than was ramipril; the adjusted hazard ratios and 95% CIs were 1.47 (95% CI, 1.14 to 1.89), 1.71 (CI, 1.29 to 2.25), 1.56 (CI, 1.13 to 2.15), 1.58 (CI, 1.10 to 2.82), and 1.28 (CI, 0.98 to 1.67), respectively. The adjusted hazard ratio associated with perindopril was 0.98 (CI, 0.60 to 1.60). The administrative databases did not contain detailed clinical information, and unmeasured factors associated with a patient's risk for death may have influenced physicians' prescription choices. Survival benefits in the first year after acute myocardial infarction in patients 65 years of age or older seem to differ according to the specific ACE inhibitor prescribed. Ramipril was associated with lower mortality than most other ACE inhibitors.

Ace revisited: a new target for structure-based drug design.

Current-generation angiotensin-converting enzyme (ACE) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of US $6 billion. However, the use of these ACE inhibitors, which were developed in the late 1970s and early 1980s, is hampered by common side effects. Moreover, we now know that ACE actually consists of two parts (called the N- and C-domains) that have different functions. Therefore, the design of specific domain-selective ACE inhibitors is expected to produce next-generation drugs that might be safer and more effective. Here we discuss the structural features of current inhibitors and outline how next-generation ACE inhibitors could be designed by using the three-dimensional molecular structure of human testis ACE. The ACE structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry.

Characterization of angiotensin-converting enzyme in canine testis

The aim of this study was to characterize angiotensin-converting enzyme (ACE) in canine testis. Detergent-extracted canine testes were sonicated in the presence of protease inhibitors and purified on an affinity column with the ACE inhibitor, lisinopril, as an affinity ligand for ACE. The fractions recovered were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based on the hydrolysis of Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8 min incubation. The specific activity of ACE in the starting testicular extracts was 3.53 +/- 0.99 mU mg(-1) protein with a 1588 times enrichment in ACE activity after lisinopril affinity chromatography (4239 +/- 2600 mU mg(-1) protein). The recovery efficiency of ACE after lisinopril affinity chromatography was 71.2%. The ACE activity in the detergent extracts and the purified fractions was inhibited significantly by 10 micromol captopril l(-1), a specific ACE inhibitor, and was restored to 88% of normal activity by the addition of the thiol-alkylating agent N-ethylmaleimide (0.5 mmol l(-1)) in the detergent extracts and the purified fractions incubated with captopril. The treatment of testicular extracts with 10 mmol EDTA l(-1) reduced the ACE activity significantly (5.40 +/- 1.26 versus 0.58 +/- 0.23 mU mg(-1)). The ACE activity was restored fully in the presence of zinc (5.28 +/- 0.70 mU mg(-1)). The anti-ACE antibody (raised against a 70 kDa protein from the periacrosomal plasma membrane of equine spermatozoa) recognized a 65-70 kDa protein in the detergent-extracted testes as well as in the affinity-purified fractions. This antibody also recognized a protein of similar molecular mass in ejaculated spermatozoa. ACE was localized in the periacrosomal area of the ejaculated spermatozoa and in spermatids in the seminiferous tubules. The results of this study demonstrate that ACE is present in canine testis and retains its enzyme activity after purification with lisinopril affinity chromatography. Activity of canine ACE is inhibited by captopril and EDTA and is restored in the presence of N-ethylmaleimide and zinc.

Characterization of angiotensin-converting enzyme in canine testis

The aim of this study was to characterize angiotensin-converting enzyme (ACE) in canine testis. Detergent-extracted canine testes were sonicated in the presence of protease inhibitors and purified on an affinity column with the ACE inhibitor, lisinopril, as an affinity ligand for ACE. The fractions recovered were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based on the hydrolysis of Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8 min incubation. The specific activity of ACE in the starting testicular extracts was 3.53 +/- 0.99 mU mg(-1) protein with a 1588 times enrichment in ACE activity after lisinopril affinity chromatography (4239 +/- 2600 mU mg(-1) protein). The recovery efficiency of ACE after lisinopril affinity chromatography was 71.2%. The ACE activity in the detergent extracts and the purified fractions was inhibited significantly by 10 micromol captopril l(-1), a specific ACE inhibitor, and was restored to 88% of normal activity by the addition of the thiol-alkylating agent N-ethylmaleimide (0.5 mmol l(-1)) in the detergent extracts and the purified fractions incubated with captopril. The treatment of testicular extracts with 10 mmol EDTA l(-1) reduced the ACE activity significantly (5.40 +/- 1.26 versus 0.58 +/- 0.23 mU mg(-1)). The ACE activity was restored fully in the presence of zinc (5.28 +/- 0.70 mU mg(-1)). The anti-ACE antibody (raised against a 70 kDa protein from the periacrosomal plasma membrane of equine spermatozoa) recognized a 65-70 kDa protein in the detergent-extracted testes as well as in the affinity-purified fractions. This antibody also recognized a protein of similar molecular mass in ejaculated spermatozoa. ACE was localized in the periacrosomal area of the ejaculated spermatozoa and in spermatids in the seminiferous tubules. The results of this study demonstrate that ACE is present in canine testis and retains its enzyme activity after purification with lisinopril affinity chromatography. Activity of canine ACE is inhibited by captopril and EDTA and is restored in the presence of N-ethylmaleimide and zinc.

In-vitro and in-vivo inhibition of rat neutral endopeptidase and angiotensin converting enzyme with the vasopeptidase inhibitor gemopatrilat.

Vasopeptidase inhibitors are single molecules that simultaneously inhibit neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE). The aim of this study was to characterize in-vitro and in-vivo inhibition of NEP and ACE in the rat with the vasopeptidase inhibitor gemopatrilat. In-vitro NEP and ACE inhibition was studied by radioinhibitory binding assay using rat renal membranes and the specific NEP inhibitor radioligand 125I-RB104 and the specific ACE inhibitor radioligand 125I-MK351A, respectively (n = 3 per curve). In-vivo NEP and ACE inhibition was studied using in-vitro autoradiography in rats that received oral gemopatrilat (1, 3, 10 mg/kg; n = 4 per dose) and were killed 1 h later, or received oral gemopatrilat (3, 10 mg/kg) and were killed at time points 1, 2, 4, 8, 18, 24 and 48 h (n = 4 per time point). Gemopatrilat caused a concentration-dependent displacement of specific radioligands from renal membrane NEP (IC50 305 +/- 5.4 nmol/I) and ACE (IC50 3.6 +/- 0.02 nmol/). In the dose-response study gemopatrilat (1, 3 and 10 mg/kg) caused significant inhibition of plasma ACE (P< 0.01), and renal ACE and NEP (3, 10 mg/kg, P < 0.01). In the time course experiment, gemopatrilat (10 mg/kg) increased plasma renin activity for 8 h (P< 0.01) and inhibited plasma ACE (P< 0.05), renal NEP (P< 0.01) and renal ACE (P< 0.05) for 48 h. Gemopatrilat is a potent in-vitro vasopeptidase inhibitor that also causes prolonged inhibition of circulating and renal ACE and renal NEP after a single oral dose. The data suggest that gemopatrilat may be a useful addition to existing vasopeptidase inhibitors in the treatment of cardiovascular disease.

Antihypertensive and antihypertrophic effects of omapatrilat in SHR

Vasopeptidase inhibitors, such as omapatrilat are single molecules that simultaneously inhibit neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE). In normotensive rats, a single dose of oral omapatrilat (10 mg/kg) and 1 mg/kg inhibited plasma ACE ( P < .01) for 24 h and increased plasma renin activity for 8 h ( P < .01). In vitro autoradiography using the specific NEP inhibitor radioligand 125I-RB104 and the specific ACE inhibitor radioligand 125I-MK351A showed omapatrilat (10 mg/kg) caused rapid and potent inhibition of renal NEP and ACE, respectively, for 24 h ( P < .01). In spontaneously hypertensive rats, 10 days of oral omapatrilat (40 mg/kg/day) reduced blood pressure (vehicle 237 ± 4 mm Hg; omapatrilat, 10 mg/kg, 212 ± 4 mm Hg; omapatrilat 40 mg/kg, 197 ± 4 mm Hg, P < .01) in a dose-dependent manner (10 v 40 mg/kg, P < .01). Left ventricular hypertrophy was significantly reduced by high-dose omapatrilat (vehicle 2.76 ± 0.03 mg/g body weight; omapatrilat, 10 mg/kg, 2.71 ± 0.02 mg/g; omapatrilat 40 mg/kg, 2.55 ± 0.02 mg/g, P < .01) and omapatrilat also increased kidney weight compared to vehicle (both doses, P < .01). Omapatrilat caused significant inhibition of plasma ACE and increased plasma renin activity (both doses, P < .01), and in vitro autoradiographic studies indicated sustained inhibition of renal ACE and NEP (both doses, P < .01). Omapatrilat is a potent vasopeptidase inhibitor, and its antihypertensive effects are associated with inhibition of NEP and ACE at the tissue level and beneficial effects on cardiovascular structure. Relating the degree of tissue inhibition to physiologic responses may allow further definition of the role of local renin angiotensin and natriuretic peptide systems in the beneficial effects of vasopeptidase inhibitors.