Angiotensin-converting enzyme and cardiovascular disease risk.

The published studies of the association of the angiotensin-converting enzyme (ACE) genotype with cardiovascular disease have used many different diagnostic criteria for cardiovascular disease and have drawn their samples from different patient groups and different populations. This review examines the association of the ACE DD genotype with cardiovascular disease risk in studies grouped by their case criterion, the geographical region of the population samples, and by the cardiovascular risk level of the patient sample. In studies where the underlying odds ratios are determined to be homogeneous, the overall odds ratios for myocardial infarction and coronary artery disease with regard to the ACE DD genotype are estimated using the Mantel-Haenszel method.     

The NH2- and COOH-terminal sequences of the angiotensin-converting enzyme isozymes from rabbit lung and testis

The NH₂-and COOH-terminal sequences of the angiotensin-converting enzymes from rabbit lung and testis have been determined using less than 0.6mg of each protein. They are: (NH₂)Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp- and -(Phe, Tyr)-Ser-Leu-Ala(COOH) for the pulmonary enzyme; and (NH₂)Arg-Arg-Val-Ser-Asn-Asn-Gln-Ser-Ser- and -(Phe, Ala)-Glu-Leu-Ser(COOH) for the enzyme from testis.     

Angiotensin-converting enzyme and anorexia nervosa

Angiotensin-converting enzyme (ACE) activity was measured in 10 patients with anorexia nervosa, 6 with hyperthyroid Graves' disease, and 7 with primary hypothyroidism. Patients with anorexia nervosa had a low serum ACE activity (9.8 +/- 2.2 IU/l), as compared to findings in normal subjects (13.4 +/- 3.5 IU/l) (P less than 0.05). Patients with hyperthyroid Graves' disease had high serum ACE activity (23.7 +/- 5.8 IU/l), as compared to levels in normal subjects (P less than 0.01), and patients with primary hypothyroidism tended to have low serum ACE activity (10.1 +/- 1.8 IU/l), compared to the normal subjects (P less than 0.1). Following weight gain (before; 71.3 +/- 10.2% of ideal body weight, after; 88.7 +/- 5.6% of ideal body weight), serum ACE activity in patients with anorexia nervosa reverted to within the normal range (13.8 +/- 3.5 IU/l), and serum T3 concentration was restored to the normal range (before; 0.7 +/- 0.2 ng/ml, after; 1.1 +/- 0.3 ng/ml). In these patients, ACE activity correlated with the per cent of ideal body weight (P less than 0.05). These data suggest that, in underweight subjects with anorexia nervosa, decreased serum ACE activities may relate to emaciation.     

Determination in plasma of angiotensin-converting enzyme inhibitor by inhibitor-binding assay

A method of determining a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite (RS-5139) in plasma by inhibitor-binding assay has been developed using high-performance liquid chromatography. The assay is based on the principle that the amount of inhibitor bound to the enzyme is inversely related to the amount of hippuric acid liberated on hydrolysis from the artificial substrate (hippuryl- -hystidyl- -leucine). Plasma was heated at 60°C for 15 min, to inactivate endogenous enzyme, and preincubated with rabbit-lung angiotensin-converting enzyme at 37°C for 3 min. The artificial substrate (5.75 mg/ml in pH 8.3 phosphate buffer containing sodium chloride) was added to the resulting solution, and the mixture was incubated for 30 min. The reaction was terminated by the addition of 2 M hydrochloric acid. The hippuric acid liberated on hydrolysis was extracted with ethyl acetate and determined by reversed-phase chromatography using methylparaben as an internal standard. The total concentration of the inhibitor and its metabolite were determined by this method after de-esterification by rat-plasma esterase. The standard curve was obtained by the regression analysis of log concentration against logit response. The within-day and day-to-day precision were satisfactory. The proposed method is simple, rapid and sensitive enough to determine angiotensin-converting enzyme inhibitor in plasma.     

Angiotensin-converting enzyme activity in ovine bronchial vasculature.

Angiotensin-converting enzyme (ACE) plays a major role in the metabolism of bradykinin, angiotensin, and neuropeptides, which are all implicated in inflammatory airway diseases. The activity of ACE, which is localized on the luminal surface of endothelial cells (EC), has been well documented in pulmonary EC; however, few data exist regarding the relative activity of ACE in the airway vasculature. Therefore, we measured ACE activity in cultured EC from the sheep bronchial artery and bronchial mucosa (microvascular) and compared it with pulmonary artery EC. The baseline level of total ACE activity (cellular plus secreted) was significantly greater in bronchial microvascular EC (1.24 +/- 0.24 mU/106 cells) compared with bronchial artery EC (0.59 +/- 0.15 mU/106 cells; P < 0.05) and comparable to pulmonary artery EC (1.12 +/- 0.14 mU/106 cells; P > 0.05). Measured ACE activity secreted into culture medium for each cell type was 64-74% of total activity and did not differ among the three EC types (P = 0.17). Hydrocortisone (10 microg/ml; 48-72 h) treatment resulted in a significant increase in ACE activity in bronchial EC. Likewise, TNF-alpha (0.1 ng/ml) treatment markedly increased ACE activity in all cell lysates (P < 0.05). We confirmed the importance of ACE activity in vivo since, at the highest dose of bradykinin studied (10-8 M), bronchial artery pressure at constant flow showed a greater decrease after captopril treatment (36% before vs. 60% after; P = 0.05). These results demonstrate high ACE expression of the bronchial microvasculature and suggest an important regulatory role for ACE in the metabolism of kinin peptides known to contribute to airway pathology.     

Angiotensin-converting enzyme activity in isolated brain microvessels.

The localization of angiotensin-converting enzyme (kininase II; ACE) in bovine cerebral cortex was studied by mechanically isolating microvessels from surrounding brain parenchyma. ACE specific activity, as assayed by generation of L-histidyl-L-leucine from the synthetic substrate hippuryl-L-histidyl-L-leucine, was enriched approximately 30 times in microvessels compared to homogenates of intact cerebral cortical gray matter. The nonapeptide 9a, SQ20,881), the orally active anti-hypertensive drug, 2-D-methyl-3-mercaptopropanoyl-L-proline (SQ14,225), and the vasoactive peptides bradykinin and angiotensin II inhibited this activity in a dose-dependent fashion. Brain microvessel ACE required chloride for optimal activity, was potentiated by cobalt nitrate, and was inhibited by the chelating agents EDTA and o-phenanthroline. Enzymatic generation of histidyl-leucine also was observed with the naturally occurring decapeptide substrate angiotensin I. In addition, microvessels obtained from bovine cerebellar cortex, hippocampus and corpus striatum, as well as from the cerebral cortex of Sprague-Dawley rats, were enriched in ACE activity. The presence of angiotensin-converting enzyme in brain microvessels suggests that cellular components of the blood-brain barrier may participate in the metabolism of peptide hormones such as angiotensin I and bradykinin within the central nervous system.     

Angiotensin-converting enzyme: accumulation in medium from cultured endothelial cells.

The activity of angiotensin converting enzyme has been measured in endothelial cells cultured from hog aorta, and in serum-free culture medium taken from both endothelial cells and smooth muscle cells. Endothelial cells maintained in medium containing 20% fetal calf serum contained 43 pmol/min/10⁶ cells of converting enzyme activity; freshly collected cells contained 518 pmol/min/10⁶ cells. Endothelial cells held in serum-free medium release this activity into the medium in amounts up to 40 times that associated with the cells; at the same time the activity associated with the cells rises 2 fold. The rise in cell-associated activity and the appearance of activity in the medium are both blocked by cycloheximide. These observations provide direct evidence that endothelial cells can produce excess angiotensin-converting enzyme and release it in active form, and thus serve as the source of circulating converting enzyme activity.     

Angiotensin-converting enzyme inhibitors: biochemical properties and biological actions

The review will cover the chemistry and biochemistry of angiotensin-converting enzyme inhibitors with emphasis on data published since the publication of previous reviews. The relative merits of each contribution will be evaluated, as well as their potential for leading to new discoveries. The biology of angiotensin-converting enzyme inhibitors will be brought up-to-date to give the reader an appreciation of the medical implications of this new type of antihypertensive agent.     

Angiotensin-converting enzyme: immunologic, structural, and developmental aspects

Immunization of dog and rat high pure rabbit pulmonary angiotensin-converting enzyme elicited, in some individuals, antibodies that inhibited their own converting enzyme. Active immunization with an immunologically related enzyme is thus a plausible approach for developing biologically based inhibitors of enzymes that are either in or accessible to the circulation. Rabbit testicular peptidyldipeptide hydrolase was purified to homogeneity and found to be a considerably smaller (Mr approximately 100,000) glycoprotein than pulmonary converting enzyme (Mr approximately 140,000). The two enzymes differed at their amino- and carboxy-termini. However, they exhibited identical catalytic properties, and antibodies prepared against either inhibited both similarly. In competition radioimmunoassays, antibodies against the pulmonary enzyme preferred it to the testicular species, whereas those against the latter did not distinguish between the two molecules. The testicular isozyme thus resembles an internal part of the pulmonary polypeptide, which includes its active site. In a reticulocyte lysate, mRNA from the lungs of immature and mature rabbits comparably primed the synthesis of a polypeptide (Mr approximately 129,000) that reacted with anticonverting enzyme antibodies. In contrast, an immunoreactive species was programed only by mRNA from the testis of mature animals, and this protein was much smaller (Mr approximately 85,000). Maturation dependence and a shorter polypeptide chain, the regulatory and structural properties that distinguish the testicular isozyme, are thus each pretranslationally determined.     

Organ-specific human alcohol dehydrogenase: isolation and characterization of isozymes from testis

Class III alcohol dehydrogenase (ADH) predominates in human testis. The two isozymes of this class were isolated jointly by affinity and conventional ion exchange chromatography. They display anodic electrophoretic mobility at pH 8.2, are completely insensitive to 4-methylpyrazole inhibition and oxidize ethanol and other short-chain primary alcohols very poorly. Thus, their kinetic and inhibition characteristics are identical to human liver class III ADH. In contrast, class I ADH is a barely detectable component of testicular alcohol dehydrogenase. The physicochemical characteristics of class III ADH are virtually identical to those of alcohol dehydrogenases found in other organs.     

Angiotensin-converting enzyme ID polymorphism and fitness phenotype in the HERITAGE Family Study.

It has been suggested that genetic variation in the angiotensin-converting enzyme (ACE) gene is associated with physical performance. We studied the association between the ACE insertion (I)/deletion (D) polymorphism and several fitness phenotypes measured before and after 20 wk of a standardized endurance training program in sedentary Caucasian (n = 476) and black (n = 248) subjects. Phenotypes measured were oxygen uptake (VO(2)), work rate, heart rate, minute ventilation, tidal volume, and blood lactate levels during maximal and submaximal [50 W and at 60 and 80% of maximal VO(2) (VO(2 max))] exercise and stroke volume and cardiac output during submaximal exercise (50 W and at 60% VO(2 max)). The ACE ID polymorphism was typed with the three-primer PCR method. Out of 216 association tests performed on 54 phenotypes in 4 groups of participants, only 11 showed significant (P values from 0.042 to 0. 0001) associations with the ACE ID polymorphism. In contrast to previous claims, in Caucasian offspring, the DD homozygotes showed a 14-38% greater increase with training in VO(2 max), VO(2) at 80% of VO(2 max), and all work rate phenotypes and a 36% greater decrease in heart rate at 50 W than did the II homozygotes. No associations were evident in Caucasian parents or black parents or offspring. Thus these data do not support the hypothesis that the ACE ID polymorphism plays a major role in cardiorespiratory endurance.     

Lipoxygenase-like enzyme in rat testis microsomes.

Microsomes, separated from rat testes, were found capable of oxidizing linoleate and arachidonate. The enzyme activity was solubilized with 1% Triton X-100 in acetate buffer (pH 5.0) and purified by affinity chromatography. The overall purification from the starting preparation was approx. 40-fold. The affinity-purified enzyme was almost homogeneous as determined by electrophoresis in polyacrylamide gel. The enzyme was characterized as lipoxygenase-like from its spectrum, specificity, effect of linoleate on its fluorescence and linoleate oxidation products. Three types of compounds separated by thin-layer chromatography were generally present in the lipoxygenase-like enzyme reaction on linoleic acid: substrate fatty acid, polar by-products and hydroperoxides. The hydroperoxides were analyzed by infrared spectra and mass spectrometry and showed the presence of both 9- and 13-hydroxy isomers.     

Angiotensin-converting enzyme signalling in human preadipocytes and adipocytes

Angiotensin-converting enzyme (ACE, kininase II) is a plasma membrane zinc metallopeptidase that acts as a key enzyme for the extracellular conversion of vasoactive peptides. Recently, ACE outside-in signalling in endothelial cells has been described. The present study tested the hypothesis that ACE signalling is not restricted to endothelial cells and may act as an additional peptide receptor on human preadipocytes and adipocytes. ACE protein levels were not changed during adipose conversion of human primary preadipocytes. The enzyme was primarily localized to the non-detergent-resistant fraction of the membrane and phosphorylated in non-dividing cells. Antibody arrays of whole cell lysate detected putative ACE-interacting proteins, which all share important roles in cell cycle control and/or apoptosis. These findings suggest that ACE is a versatile molecule, involved both in the regulation of extracellular peptide concentrations and direct intracellular signalling. In human adipose cells ACE may potentially influence exit from the cell cycle, differentiation, and programmed cell death signalling.     

Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: determinants and constraints

ADAM17, also known as tumor necrosis factor α-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) → Glu(708) mutation and the Arg(708)Arg(710) → Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.