Regulation of angiotensin I-converting enzyme in cultured bovine bronchial epithelial cells

The purpose of this study was to determine whether angiotensin I-converting enzyme (ACE) is present in cultured bovine bronchial epithelial cells (BBECs) and whether its activity can be modulated. We found that extracts of confluent monolayers of cultured BBECs degraded [glycine-1-¹⁴C]hippuryl-L -histidyl-L -leucine at a rate of 843 ± 66 pmol/hr/mg protein (mean ± SEM, n = 5). In addition, we found that the enzyme was shed into the culture medium. ACE activity in BBECs was inhibited by three selective, but structurally different, ACE inhibitors (captopril, quinapril, and cisalaprilat) with an IC₅₀ of approximately 2 nM. Increasing chloride concentration in the assay buffer resulted in an increase in BBECs ACE activity of 63%. Enzyme activity was also modulated by the presence of zinc cation in the assay buffer. Addition of dexamethasone to the culture medium was associated with a significant increase in BBECs ACE activity (P < 0.05), which was inhibited by the steroid receptor antagonist RU 38486. Western blot analysis of BBECs, tracheal and bronchial mucosal strips utilizing a cross-reacting rabbit anti-mouse ACE antibody, showed a faint 175 kDa band and additional strong 52 kDa and 47 kDa band. The mechanism of generation of the low M.W. bands is unknown. Our data indicate the presence of ACE in cultured BBECs and that enzyme activity can be modulated.     

Chronobiologic Evaluation of Angiotensin-Converting Enzyme Activity in Serum and Lung Tissue from Normotensive and Spontaneously Hypertensive Rats

Angiotensin-converting enzyme (ACE) activity in serum and lung tissue from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was determined at six different circadian times. In WKY rats serum ACE varied significantly within 24 h, mainly due to reduced enzyme activity at 12:00 h. In SHR the 24-h profile of serum ACE did not exhibit time-dependent differences. Mean serum ACE activity over 24 h was significantly higher in WKY than in SHR. In lung tissue ACE activity did not depend on the circadian time in either strain. Mean enzyme activity in lung tissue was not different between WKY and SHR. We conclude that circadian changes in the activity of serum and tissue ACE are unlikely to play an important role in the regulation of the circadian blood pressure profile in both normotensive and spontaneously hypertensive rats.     

Chronobiologic Evaluation of Angiotensin-Converting Enzyme Activity in Serum and Lung Tissue from Normotensive and Spontaneously Hypertensive Rats

Angiotensin-converting enzyme (ACE) activity in serum and lung tissue from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was determined at six different circadian times. In WKY rats serum ACE varied significantly within 24 h, mainly due to reduced enzyme activity at 12:00 h. In SHR the 24-h profile of serum ACE did not exhibit time-dependent differences. Mean serum ACE activity over 24 h was significantly higher in WKY than in SHR. In lung tissue ACE activity did not depend on the circadian time in either strain. Mean enzyme activity in lung tissue was not different between WKY and SHR. We conclude that circadian changes in the activity of serum and tissue ACE are unlikely to play an important role in the regulation of the circadian blood pressure profile in both normotensive and spontaneously hypertensive rats.     

Serum angiotensin-converting enzyme in diabetes mellitus: a negative report

Serum angiotensin-converting enzyme (ACE) was measured by a direct photometric method in 78 normotensive diabetic patients and in 34 controls. For comparison, ACE was also assayed in 24 subjects by a radiometric procedure. We found no ACE elevations in diabetics and no significant difference in mean ACE levels between diabetics and normals. Within the diabetic group, enzyme levels were not affected by duration of disease, degree of metabolic control, or presence or not of microangiopathy. Only type I subjects had mean ACE significantly (p less than 0.05) higher than type II, very likely due to their younger age. Serum ACE data from photometric and radiometric methods significantly correlated. ACE measurement seems to be of scarce significance in diabetes mellitus.     

Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.     

Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides

Angiotensin-converting enzyme (ACE) plays a major role in the regulation of blood pressure. A diagnostic assay to measure angiotensin-converting enzyme (ACE) activity was transformed into an enzyme inhibition assay and optimised, which led to a more sensitive and less expensive assay. By this spectrophotometric method, ACE inhibition is measured using the substrate furanacryloyl-Phe-Gly-Gly and as ACE source rabbit lung acetone extract. The optimised as well as the original ACE inhibition assay were used to verify the ACE inhibitory activity of captopril. The ACE inhibition assay was further validated by enalapril, its active derivative enalaprilat and the ACE-inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, corresponding to a tryptic fragment of bovine beta-lactoglobulin. Sigmoid curves could be fit adequately to the data points representing ACE inhibition in function of inhibitor concentration. IC(50) values for these compounds corresponded well with literature data. Furthermore, pea and whey protein hydrolysates obtained by digestion with trypsin showed ACE inhibitory activity in the ACE inhibition assay. Hence, this optimised assay is suitable to screen for ACE inhibitory peptides derived from food proteins with a possible antihypertensive effect in vivo.     

A fluorometric assay for angiotensin-converting enzyme activity

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.     

Structural organization of membrane and soluble forms of somatic angiotensin-converting enzyme.

The catalytic activity and quaternary structure of soluble (s) and membrane (m) forms of angiotensin-converting enzyme (ACE) were studied in reversed micelles of ternary system Aerosol OT--water--octane. The profile of the dependence of the catalytic activity of the two enzyme forms on the degree of surfactant hydration (micellar size) had several optima corresponding to the function of various active oligomeric enzyme forms; the curves for the s- and m-forms of ACE were different. Data of sedimentation analysis prove that in reversed micelles, s-ACE can exist as monomers, dimers, or tetramers depending on the hydration degree, and the m-form is present as dimers and tetramers only. The values of the kinetic parameters for the hydrolysis of the substrate furylacryloyl-Phe-Gly-Gly by all the enzyme forms were determined, and the data indicate that the activity of the m-form is enhanced by oligomerization. The ACE activity strongly depends on the medium; it is higher when ACE is in contact with matrix or other enzyme molecules.     

Release of angiotensin I-converting enzyme by endothelial cells in vitro

Bovine fetal aortic endothelial cells cultured in serum-containing medium accumulate angiotensin I-converting enzyme (ACE) activity and also release it into the culture medium. Following subcultivation of a confluent culture using trypsin-EDTA, cellular ACE activity falls 50% within 8 h, but no ACE activity is detected in the medium, suggesting intracellular loss of the enzyme activity. ACE activity reappears in both the cell lysate and culture medium after the culture becomes confluent. The rate of accumulation of ACE activity released into the medium is always greater than that for cellular activity. For example, 21 days following subcultivation 80-85% of the total culture activity is detected in the medium. Both cellular and medium-associated ACE decrease proportionately as the culture progresses through its in vitro lifespan.     

Modulation of glucose transporters in rat diaphragm by sodium tungstate

The somatic isoform of angiotensin-converting enzyme (ACE) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. We have used the two-domain ACE form and its individual domains to compare characteristics of different domains and to probe mutual functioning of the two active sites within a bovine ACE molecule. The substrate Cbz-Phe-His-Leu (N-carbobenzoxy-L-phenylalanyl-L-histidyl-L-leucine; from the panel of seven) was hydrolyzed faster by the N-domain, the substrates FA-Phe-Gly-Gly (N-(3-[2-furyl]acryloyl)-L-phenylalanyl-glycyl-glycine) and Hip-His-Leu (N-benzoyl-glycyl-L-histidyl-L-leucine) were hydrolyzed by both domains with equal rates, while other substrates were preferentially hydrolyzed by the C-domain. The inhibitor captopril ((2S)-1-(3-mercapto-2-methylpropionyl)-L-proline) bound to the N-domain more effectively than to the C-domain, whereas lisinopril ((S)-N(alpha)-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) bound to equal extent with all ACE forms. However, active site titration with lisinopril assayed by hydrolysis of FA-Phe-Gly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either two-domain or single-domain ACE forms, indicating that a single active site functions in bovine somatic ACE. Neither of the k(cat) values obtained for somatic enzyme was the sum of k(cat) values for individual domains, but in every case the value of the catalytic constant of the hydrolysis of the substrate by the two-domain ACE represented the mean quantity of the values of the corresponding catalytic constants obtained for single-domain forms. The results indicate that the two active sites within bovine somatic ACE exhibit strong negative cooperativity.     

Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks

Oligopeptides of 1 KDa or less were obtained by hydrolysis of chicken egg yolks with a crude enzyme, and by dialysis with a semipermeable membrane filter. Since the extracted peptides had an inhibitory action on the activity of angiotensin I-converting enzyme (ACE) in vitro, they were orally administered at 20, 100 and 500 mg/kg body weight to spontaneously hypertensive rats (SHR) for 12 weeks to analyze the physiological role on cardiovascular functions. The administered oligopeptides suppressed the development of hypertension at all dosages. After 12 weeks at 500 mg/kg body weight, the values for systolic, mean, and diastolic blood pressure were approximately 10% less in SHRs administered than controls. Furthermore, serum ACE activity of the peptide-administered groups was significantly lower than that of the control group in a dose-related manner. Our results imply that oligopeptides extracted from hen's egg yolks could potentially suppress the development of hypertension in SHR, and this effect might be induced by the inhibition of ACE activity.     

SARS病毒受体ACE2的克隆、原核表达及其功能区鉴定

ACE2(angiotensin-converting enzyme 2,ACE2)是SARS冠状病毒(severe acute respiratory syndrome associatedcoronavirus,SARS-CoV)的主要受体。此研究旨在鉴定ACE2的SARS-CoV受体功能区,为进一步阐明SARS-CoV与细胞间的相互作用机制及研制抗病毒药物等提供理论依据。利用RT-PCR从Vero-E6细胞的mRNA中分两段扩增ACE2基因,其中N端片段ACE2A1-367(102~1 210nt)不包括ACE2的酶活性位点(1 223~1 237nt,或374~378aa),而C端片段ACE2B335-805(1 101~2 524nt)包括ACE2的酶活性位点。扩增片段克隆入pMD-18T,并进行测序鉴定。进一步构建与GST基因融合表达的原核表达质粒pGEX-ACE2A与pGEX-ACE2B,IPTG诱导表达。表达的融合蛋白分子量为65kD和77kD,主要以包涵体形式存在。Western blot证实表达产物具有免疫学活性。将纯化的包涵体蛋白质复性后进行Western blot分析,证实pGEX-ACE2A表达的蛋白(~65kD)能与SARS-CoV S1蛋白特异结合,而pGEX-ACE2B表达的蛋白(~77kD)不能与S1蛋白结合。结果表明,ACE2的受体活性与酶活性位点无关,受体功能区在ACE2 N端367个氨基酸内。     

Sex Hormones Promote Opposite Effects on ACE and ACE2 Activity,Hypertrophy and Cardiac Contractility in Spontaneously Hypertensive Rats

BackgroundThere is growing interest in sex differences and RAS components. However, whether gender influences cardiac angiotensin I-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity is still unknown. In the present work, we determined the relationship between ACE and ACE2 activity, left ventricular function and gender in spontaneously hypertensive rats (SHRs).ConclusionOvariectomy leads to increased cardiac hypertrophy, ACE2 activity, PLB expression and PLB to SERCA2a ratio, and worsening of hemodynamic variables, whereas in males the removal of testosterone has the opposite effects on RAS components.     

Different distribution of two types of angiotensin II-generating enzymes in the aortic wall

Dog, monkey and human aortic tissues contained two distinct types of angiotensin II-generating enzymes; angiotensin converting enzyme (ACE) and chymostatin-sensitive angiotensin II-generating enzyme (CAGE). Endothelium, media and adventitia of canine thoracic aortae were separated using collagenase digestion, and determined for their ACE and CAGE activity. ACE activity was assayed by hippuryl-His-Leu cleavage. CAGE activity was estimated with ANG I as substrate in the presence of inhibitors of ACE and angiotensinases. His-Leu, the common product of both enzyme reactions, was fluorimetrically quantified after o-phthalaldehyde condensation. ACE localized mainly in endothelium, while CAGE distributed predominantly in adventitia. Similar results were obtained with human and monkey aortae. Such a contrasting distribution may indicate the distinct functional role of these two enzymes.     

Unusual Behavior of Membrane Somatic Angiotensin-Converting Enzyme in a Reversed Micelle System

Properties of the membrane and soluble forms of somatic angiotensin-converting enzyme (ACE) were studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. The membrane enzyme with a hydrophobic peptide anchor was more sensitive to anions and to changes in pH and composition of the medium than the soluble enzyme without anchor. The activity of both forms of the enzyme in the reversed micelles significantly depended on the molarity of the buffer added to the medium (Mes-Tris-buffer, 50 mM NaCl). The maximum activity of the soluble ACE was recorded at buffer concentration of 20-50 mM, whereas the membrane enzyme was most active at 2-10 mM buffer. At buffer concentrations above 20 mM, the rate of hydrolysis of the substrate furylacryloyl-L-phenylalanyl-glycylglycine by both ACE forms was maximal at pH 7.5 both in the reversed micelles and in aqueous solutions. However, at lower concentrations of the buffer (2-10 mM), the membrane enzyme had activity optimum at pH 5.5. Therefore, it is suggested that two conformers of the membrane ACE with differing pH optima for activity and limiting values of catalytic constants should exist in the reversed micelle system with various medium compositions. The data suggest that the activity of the membrane-bound somatic ACE can be regulated by changes in the microenvironment.     

Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides

An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture.     

Increased urinary angiotensin-converting enzyme 2 in renal transplant patients with diabetes

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n?=?100) and healthy controls (age 45 yrs, 26% male, n?=?50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p?=?0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p?=?0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients.     

Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research

Cell culture experiments often employ the use of culture media that contain fetal calf serum (FCS). The angiotensin peptides angiotensin II and angiotensin 1–7 have opposing effects with angiotensin converting enzyme 2 (ACE2) being the enzyme predominantly responsible for generating angiotensin 1–7 from angiotensin II. The effect of FCS on angiotensin peptides has not previously been described. We have shown that FCS has ACE2 enzyme activity capable of degrading angiotensin II and generating angiotensin 1–7. Researchers should be aware that FCS possesses ACE2 activity and that heat-treating FCS to 56 °C only partially inhibits this enzyme activity, whereas heat-treating to 70 °C completely abolishes ACE2 activity.     

Plasma angiotensin-converting enzyme activity and blood pressure during the first year of life in normotensive and spontaneously hypertensive rat.

Plasma angiotensin-converting enzyme (ACE) activity and systolic blood pressure were studied every consecutive month during the first years of life in male spontaneously hypertensive (SHR) and in normotensive rats (NWR). During the first month after birth neither ACE activity nor systolic blood pressure showed significant difference between SHR and NWR. ACE activity in SHR was significantly reduced from 2nd till 12th month of age in comparison with age-matched NWR. In the 2nd month of age the systolic blood pressure was significantly higher in SHR than in NWR, it increased further until the 5th month and was maintained at this high level till the 12th month. No correlation between changes in the systolic blood pressure and the ACE activity was found in SHR after the 2nd month of age. During the investigation period no age-related dynamics of ACE activity was observed in SHR. The observed difference of ACE activity was not due to an increase of plasma his-leu hydrolyzing activity in SHR and was not abolished after a 24-hour dialysis of plasma. This difference could not be caused by the altered effect of chloride ion on the enzyme since similar pattern of Cl-dependent activation of plasma ACE in 4-month-old SHR and NWR was observed. Lineweaver-Burke plot analysis revealed that this difference appears to be due to a decrease of the enzyme maximal velocity in SHR but to a change of the Km value of ACE for the substrate hippuryl-1-his-1-leu. Our data provide evidence for a lower concentration of the available active enzyme molecules in SHR plasma in respect to NWR after the 1st month of life. Whether the reduced ACE activity in SHR is a consequence of the increased blood pressure remains to be elucidated.     

Inhibition mechanism and model of an angiotensin I-converting enzyme (ACE)-inhibitory hexapeptide from yeast (Saccharomyces cerevisiae)

Angiotensin I-converting enzyme (ACE) has an important function in blood pressure regulation. ACE-inhibitory peptides can lower blood pressure by inhibiting ACE activity. Based on the sequence of an ACE-inhibitory hexapeptide (TPTQQS) purified from yeast, enzyme kinetics experiments, isothermal titration calorimetry (ITC), and a docking simulation were performed. The hexapeptide was found to inhibit ACE in a non-competitive manner, as supported by the structural model. The hexapeptide bound to ACE via interactions of the N-terminal Thr1, Thr3, and Gln4 residues with the residues on the lid structure of ACE, and the C-terminal Ser6 attracted the zinc ion, which is vital for ACE catalysis. The displacement of the zinc ion from the active site resulted in the inhibition of ACE activity. The structural model based on the docking simulation was supported by experiments in which the peptide was modified. This study provides a new inhibitory mechanism of ACE by a peptide which broads our knowledge for drug designing against enzyme targets.