Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Summary Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.     

Isolation of human liver angiotensin-converting enzyme by chromatofocusing

Angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human liver by chromatofocusing. The isolation procedure permitted us to obtain a 9000-fold purified enzyme with a 22% yield. Specific activity of the angiotensin-converting enzyme was 10 units/mg of protein. The molecular mass of enzyme determined by polyacrylamide gel electrophoresis under denaturing conditions was 150,000. The isoelectric point (4.2-4.3) was also determined by chromatofocusing. The Km values of the enzyme for hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine are 5000 and 125 microM, respectively. The human liver angiotensin-converting enzyme is inhibited by bradykinin-potentiating factor SQ 20881 (IC50 = 18 nM).     

Formation of angiotensin III by angiotensin-converting enzyme.

Angiotensin III is formed from des-Asp1 -angiotensin I by angiotensin-converting enzyme. The Km (11 muM) of the reaction is one-third of that for the conversion of angiotensin I into angiotensin II. As suggested by the Km values, bradykinin, peptide BPP9a and angiotensins II and III are better inhibitors of the formation of angiotensin II than of the formation of angiotensin III.     

Inhibition of angiotensin-converting enzyme by des-Leu10-angiotensin I: a potential mechanism of endogenous angiotensin-converting enzyme regulation

Des-Leu10-angiotensin I is a nonapeptide generated from angiotensin I by the action of carboxypeptidase-like activities residing in the human platelet and mast cell. This nonapeptide was found to inhibit rabbit lung angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) with a Ki of 3.1 X 10(-7) M. The mechanism of inhibition was competitive. Inhibition of human serum angiotensin-converting enzyme by des-Leu10-angiotensin I was comparable in magnitude to inhibition by bradykinin and angiotensin III. These results suggest that limited proteolysis of angiotensin I by cells resident in vascular tissue may result in the generation of an endogenous inhibitor of angiotensin-converting enzyme. Such pathways may play roles in controlling levels of vasoactive peptides at local vascular sites.     

Evolution of angiotensin-converting enzyme inhibitors

The renin-angiotensin system is perhaps the most important hormonal system in the regulation of blood pressure. Its influence on blood pressure is mediated by the potent vasoconstrictor angiotensin II. Since angiotensin-converting enzyme performs the last step in the biosynthesis of angiotensin II, inhibition of this enzyme has attracted the attention of many researchers as a novel approach in the control of high blood pressure. The evolution of inhibitors of this enzyme will be traced from the early snake venom peptide inhibitors to the drugs currently available for the treatment of high blood pressure and congestive heart failure.     

Structural diversity of angiotensin-converting enzyme

The crystal structure of a Drosophila angiotensin-converting enzyme (ANCE) has recently been solved, revealing features important for the binding of ACE inhibitors and allowing molecular comparisons with the structure of human testicular angiotensin-converting enzyme (tACE). ACER is a second Drosophila ACE that displays both common and distinctive properties. Here we report further functional differences between ANCE and ACER and have constructed a homology model of ACER to help explain these. The model predicts a lack of the Cl(-)-binding sites, and therefore the strong activation of ACER activity towards enkephalinamide peptides by NaCl suggests alternative sites for Cl(-) binding. There is a marked difference in the electrostatic charge of the substrate channel between ANCE and ACER, which may explain why the electropositive peptide, MKRSRGPSPRR, is cleaved efficiently by ANCE with a low K(m), but does not bind to ACER. Bradykinin (BK) peptides are excellent ANCE substrates. Models of BK docked in the substrate channel suggest that the peptide adopts an N-terminal beta-turn, permitting a tight fit of the peptide in the substrate channel. This, together with ionic interactions between the guanidino group of Arg9 of BK and the side chains of Asp360 and Glu150 in the S(2)' pocket, are possible reasons for the high-affinity binding of BK. The replacement of Asp360 with a histidine in ACER would explain the higher K(m) recorded for the hydrolysis of BK peptides by this enzyme. Other differences in the S(2)' site of ANCE and ACER also explain the selectivity of RXPA380, a selective inhibitor of human C-domain ACE, which also preferentially inhibits ACER. These structural and enzymatic studies provide insight into the molecular basis for the distinctive enzymatic features of ANCE and ACER.     

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.     

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.     

Function of angiotensin-converting enzyme on matrices

The binding of the angiotensin-converting enzyme from bovine lung on BrCN-activated Sepharose, CH- and AH-Sepharoses as well as on AH-Sepharose via the carbohydrate fragment of the glycoprotein molecule modulates the possible microenvironment of the enzyme in vivo. It has been shown that the close interaction of the enzyme with the carbohydrate matrix may increase the absolute values of catalytic constants for the hydrolysis of certain substrates. The binding of the angiotensin-converting enzyme to the matrices markedly changes the enzyme activation by chloride ions by causing a shift in the activity optima towards lower activator concentrations.     

Mechanism of increased angiotensin-converting enzyme activity stimulated by platelet-activating factor

We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 x 10(-10) to 1 x 10(-6) M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 x 10(-6) M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca2+ influx and inositol 1,4,5-trisphosphate (IP3) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP3. To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca2+ influx and IP3 release stimulated by 10(-6) M of PAF were suppressed by about 60-70%. ACE activity was also inhibited up to 70% in the presence of PAF (10(-10) - 10(-6) M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activity in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP3-mediated Ca2+ release from intracellular stores.     

The isolation of angiotensin-converting enzyme cDNA

Angiotensin-converting enzyme (ACE) is an Zn(II)-containing dipeptidyl carboxypeptidase that converts angiotensin I to the potent vasoconstrictor, angiotensin II. Using oligonucleotide probes based on the amino acid sequence of mouse kidney ACE, cDNA encoding this protein has been isolated. One cDNA, ACE.31, encodes the N-terminal 332 amino acids of mouse ACE, a portion of the protein containing a putative 34-amino acid leader sequence and the N terminus of the mature protein. Northern analyses with cloned ACE cDNA revealed that both mouse kidney and lung express two ACE mRNAs, one of 4900 and another of 4150 bases. Southern analysis suggests that cDNA ACE.31 is the product of a single gene, and thus these data add evidence to the hypothesis that the converting enzymes produced by epithelial and endothelial cells are identical.     

Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].     

Oxidative inactivation of angiotensin-converting enzyme]

Hydrogen peroxide inactivates the purified human angiotensin-converting enzyme (ACE) in vitro; the inactivating effect of H2O2 is eliminated by an addition of catalase. The lung and kidney ACE are equally sensitive to the effect of hydrogen peroxide. After addition of oxidants (H2O2 alone or H2O2 + ascorbate or H2O2 + Fe2+ mixtures) to the membranes or homogenates of the lung, the inactivation of membrane-bound ACE is far less pronounced despite the large-scale accumulation of lipid peroxidation products. The marked inactivation of ACE in the membrane fraction (up to 55% of original activity) was observed during ACE incubation with a glucose:glucose oxidase:Fe2+ mixture. Presumably the oxidative potential of H2O2 in tissues in consumed, predominantly, for the oxidation of other components of the membrane (e.g., lipids) rather than for ACE inactivation.     

Pulmonary angiotensin-converting enzyme antienzyme antibody.

A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay.     

Synergistic binding of ligands to angiotensin-converting enzyme

We have investigated the interaction of ligands in the active site of the angiotensin-converting enzyme from rabbit lung by monitoring the concurrent effects of two inhibitors on enzyme activity. A strong synergism is found in the binding of N-acetyl-L-proline (an analog of the COOH-terminal dipeptide portion of preferred substrates) and acetohydroxamate (a zinc ligand). Analysis of the inhibition data with the Yone-tani-Theorell plot yields an unusually low value of 0.0063 for the interaction constant (alpha). This result indicates that each of the above ligands stimulates the binding of the other by about 150-fold. Similar but often less pronounced synergism is observed for other zinc ligands and with some other N-acyl amino acids. These specific structural requirements suggest that the above effect is associated with an induced-fit mechanism which brings the important zinc atom into a catalytically optimal state only in the presence of certain preferred substrates.     

Induction of angiotensin-converting enzyme by oncostatin m in human endothelial cells

OBJECTIVE: To examine the role of oncostatin M (OSM) in the regulation of angiotensin converting enzyme (ACE) in endothelial cells. METHODS: Cultured endothelial cells were incubated with OSM (25-200 pM) for 24 h. Incubations were performed without or with the tyrosine kinase inhibitor, herbimycin (87 nM), or the selective MAP kinase kinase inhibitor, PD98059 (50 microM). ACE amount in intact endothelial cells was measured by an inhibitor binding assay and ACE mRNA levels by RNase protection assay. RESULTS: OSM caused a dose dependent increase in ACE amount and increased the expression of ACE mRNA. The stimulatory effect of OSM was inhibited by pretreatments with herbimycin or PD98059. CONCLUSIONS: OSM induced ACE in cultured HUVECs. Tyrosine kinase and MAPK activation were probably involved in ACE induction. Local induction of ACE by OSM in the vascular wall may be a consequence of inflammatory processes leading to locally increased production of angiotensin II and breakdown of bradykinin.     

Regulation of angiotensin-converting enzyme production by nicotine in human endothelial cells

Nicotine, a component of cigarette smoke, has been implicated in the pathogenesis of cardiovascular disease. We examined whether nicotine regulates angiotensin-converting enzyme (ACE), an enzyme that plays an important role in the pathophysiology of atherosclerosis and hypertension. Human umbilical cord vein endothelial cells were treated with nicotine (0.1-1 microM) alone or in combination with vascular endothelial growth factor (VEGF; 0.5 nM) or GF-109203X (GFX; 2.5 microM). The amount of ACE in intact endothelial cells was measured by an inhibitor-binding assay method, and ACE mRNA levels were quantified using LightCycler technology. Phosphorylated PKC levels were measured by Western immunoblotting. Nicotine did not modulate basal ACE production but significantly potentiated VEGF-induced ACE upregulation. Treatment of endothelial cells with the PKC inhibitor GFX totally blocked VEGF- and nicotine-induced ACE upregulation. VEGF induced PKC phosphorylation, which was potentiated by cotreatment with nicotine. We conclude that nicotine significantly potentiated VEGF-induced ACE upregulation. This effect was probably mediated by PKC phosphorylation. The interaction of nicotine with VEGF in ACE induction may contribute to the pathogenesis of smoking-related cardiovascular disease.