Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Summary Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells. Presented in part at the 31st Annual Meeting of the Histochemical Society, April 11–15, 1980, New Orleans, Louisiana. Wendy Baur and Ms. Jane Aghajanian for expert assistance in the preparation of the cell cultures. This work was supported by Research Grant HL 14456 and Training Grant HL 07053 from the National Institutes of Health, Bethesda, MD.     

Angiotensin I-converting enzyme in isolated human glomeruli

Angiotensin I-converting enzyme (ACE) activity was measured with hippurylhistidylleucine as a substrate in isolated human glomeruli. The mean level was 2.2 +/- 0.47 mIU/mg glomerular protein. S9780, a newly designed competitive inhibitor of ACE, inhibited this activity by 85% at 0.3 microM. [3H]S9780 specifically bound to isolated human glomeruli. The Kd value and the number of sites were 23 nM and 83 fmol/mg, respectively. The prodrug, S9490, and Captopril were less potent than S9780 in displacing [3H]S9780 from its binding sites. Angiotensin I had no effect. Binding of [3H]S9780 was inhibited after preincubation of the glomeruli with a specific polyclonal anti-human ACE antibody. These results demonstrate that ACE is present in human adult glomeruli.     

Angiotensin I-converting enzyme inhibitory peptides isolated from tofuyo fermented soybean food

Angiotensin I-converting enzyme (ACE) inhibitory activity was observed in a tofuyo (fermented soybean food) extract with an IC(50) value of 1.77 mg/ml. Two ACE inhibitors were isolated to homogeneity from the extract by adsorption and gel filtration column chromatography, and by reverse-phase high-performance liquid chromatography (HPLC). The purified substances reacted with 2,4,6-trinitrobenzensulfonic acid sodium salt. The amino acid sequences of these inhibitors determined by Edman degradation were Ile-Phe-Leu (IC(50), 44.8 microM) and Trp-Leu (IC(50), 29.9 microM). The Ile-Phe-Leu sequence is found in the alpha- and beta-subunits of beta-conglycinin, while the Trp-Leu sequence is in the B-, B1A- and BX-subunits of glycinin from soybean. Both of the peptides are non-competitive inhibitors. The inhibitory activity of Trp-Leu was completely preserved after a treatment with pepsin, chymotrypsin or trypsin. Even after successive digestion by these gastrointestinal proteases, the activity remained at 29% of the original value.     

Angiotensin I converting enzyme in gastric mucosa of the rabbit: localization by autoradiography, immunofluorescence, and immunoelectron microscopy.

To localize angiotensin converting enzyme (ACE) in the fundic mucosa of the rabbit, we used autoradiography with the ACE inhibitor [3H]-trandolaprilate and post-embedding immunocytochemical techniques with a goat anti-rabbit ACE, using fluorescence and electron microscopy. Autoradiographic localization of [3H]-trandolaprilate in rabbit fundus sections shows that ACE is present in the fundic mucosa and mainly in the gland area. Fundic mucosa was fixed with 4% formaldehyde and embedded in Lowicryl K4M. Semi-thin (1 micron) or thin sections (800-900 A) were stained with anti-rabbit ACE followed by fluorescein isothyocyanate-labeled rabbit anti-goat IgG or protein A-gold reagent, respectively. Label was present on endothelium of all blood vessels running through the mucosa. Label was prominently localized in the granules of mucous surface and neck cells and on the granules of chief cells. The intracellular localization of ACE, and particularly its intragranular presence within chief and mucous cells, suggests that the enzyme, at the fundic level, is involved in the intragranular processing of a peptide, the nature of which remains to be determined.     

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.     

Association of urinary N-domain Angiotensin I-converting enzyme with plasma inflammatory markers and endothelial function

The aim of this study was to investigate the association between urinary 90 kDa N-domain Angiotensin I-converting enzyme (ACE) form with C-reactive protein (CRP) and homocysteine plasma levels (Hcy), urinary nitric oxide (NOu), and endothelial function (EF) in normotensive subjects. Forty healthy subjects were evaluated through brachial Doppler US to test the response to reactive hyperemia and a panel of blood tests to determine CRP and Hcy levels, NOu, and urinary ACE. They were divided into groups according to the presence (ACE90+) or absence (ACE90-) of the 90 kDa ACE, the presence (FH+) or absence (FH-) of family history of hypertension, and the presence or absence of these two variables FH+/ACE90+ and FH-/ACE90-. We found an impaired endothelial dilatation in subjects who presented the 90 kDa N-domain ACE as follows: 11.4% +/- 5.3% in ACE90+ compared with 17.6% +/- 7.1% in ACE90- group and 12.4% +/- 5.6% in FH+/ACE90+ compared with 17.7% +/- 6.2% in FH-/ACE90- group, P < 0.05. Hcy and CRP levels were statistically significantly lower in FH+/ACE90+ than in FH-/ACE90- group, as follows: 10.0 +/- 2.3 microM compared with 12.7 +/- 1.5 microM, and 1.3 +/- 1.8 mg/L compared with 3.6 +/- 2.0 mg/L, respectively. A correlation between flow-mediated dilatation (FMD) and CRP, Hcy, and NOu levels was not found. Our study suggests a reduction in the basal NO production confirmed by NOu analysis in subjects with the 90 kDa N-domain ACE isoform alone or associated with a family history of hypertension. Our data suggest that the presence of the 90 kDa N-domain ACE itself may have a negative impact on flow-mediated dilatation stimulated by reactive hyperemia.     

Novel Angiotensin I-converting enzyme inhibitory peptides found in a thermolysin-treated elastin with antihypertensive activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 μg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 μg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 μM against ACE and may have potential use as a functional food.     

Dynamic aspects of alpha-actinin in cultured rabbit fibroblasts studied with immunofluorescence

In this work we study alpha-actinin sites in rabbit fibroblasts in culture. For this purpose we extracted alpha-actinin from rabbit striated skeletal muscle and produced the relative antibodies in sheep. Electrophoresis was performed, on PAA slab-gel and the immunodiffusion test on agarose slab-gel. Antibodies were used for direct and indirect immunofluorescence techniques by TRIC and FITC. We noticed that in young fibroblasts alpha-actinin is concentrated in perinuclear regions, while in adult fibroblasts it is scattered, more or less evenly in the cytoplasm and above all on the plates which link the cell membrane to the substractum. Both the direct immunofluorescence method with antibodies anti-alpha-actinin, marked by TRITC, and the indirect one with sheep IgG, marked by FITC, gave identical results.     

Ultrastructural immunocytochemical localization of clathrin in cultured fibroblasts

Using an affinity-purified antibody prepared against the major coat protein of brain-coated vesicles, clathrin, we have localized this protein by ultrastructural immunocytochemistry in Swiss 3T3 cultured fibroblasts. Fixation, permeabilization, and labeling were performed using the EGS and ferritin bridge labeling techniques. Localization of clathrin was detected on the coated regions of both the plasma membrane and the GERL apparatus. Almost no clathrin was found in the cytosol or in association with any other organelles. A very low concentration of labeling was occasionally seen randomly distributed on the inner surface of the plasma membrane and reticular membranous structures near the plasma membrane. The significance of these results in the role of the clathrin-coated regions in receptor-mediated endocytosis and Golgi function are discussed.     

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III, and bradykinin (BK) and converted ANG I to the active metabolite ANG(1–7). ANG III was converted to the novel ANG IV metabolite [des-Arg¹]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.     

Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII

Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.     

Cellular localization of neuraminidases in cultured human fibroblasts.

The cellular localization of glycoprotein and ganglioside sialidases in normal and I-cell-disease cultured fibroblasts has been investigated. Cellular organelles have been separated on a colloidal silica gradient. The subcellular distribution of these enzymes indicated that the glycoprotein sialidase is mainly a lysosomal hydrolase, whereas the ganglioside sialidase is primarily located in the plasma membranes. The latter isoenzymes is tightly bound to these membranes and thus could not be extracted by homogenization in the presence of Triton X-100. The interpretation of this finding and its relation to the pathochemistry of sialidase-deficient disorders is discussed.     

Angiotensin I-converting enzyme transition state stabilization by HIS1089: evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His(1089) as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His(1089) to Ala or Leu. At pH 7.5, with Ang I as substrate, k(cat)/K(m) values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k(cat)) with minor effects on ground state substrate binding (K(m)). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His(1089) --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His(1089) side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His(1089) on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.     

Angiotensin I-converting enzyme (ACE) activity and expression in rat central nervous system after sleep deprivation

Proteases are essential either for the release of neuropeptides from active or inactive proteins or for their inactivation. Neuropeptides have a fundamental role in sleep-wake cycle regulation and their actions are also likely to be regulated by proteolytic processing. Using fluorescence resonance energy transfer substrates, specific protease inhibitors and real-time PCR we demonstrate changes in angiotensin I-converting enzyme (ACE) expression and proteolytic activity in the central nervous system in an animal model of paradoxical sleep deprivation during 96 h (PSD). Male rats were distributed into five groups (PSD, 24 h, 48 h and 96 h of sleep recovery after PSD and control). ACE activity and mRNA levels were measured in hypothalamus, hippocampus, brainstem, cerebral cortex and striatum tissue extracts. In the hypothalamus, the significant decrease in activity and mRNA levels, after PSD, was only totally reversed after 96 h of sleep recovery. In the brainstem and hippocampus, although significant, changes in mRNA do not parallel changes in ACE specific activity. Changes in ACE activity could affect angiotensin II generation, angiotensin 1-7, bradykinin and opioid peptides metabolism. ACE expression and activity modifications are likely related to some of the physiological changes (cardiovascular, stress, cognition, metabolism function, water and energy balance) observed during and after sleep deprivation.     

Histone localization in polytene chromosomes by immunofluorescence

Polytene chromosomes of Chironomus thummi were treated with antisera elicited by purified calf thymus histone fractions, and the location of each histone type was visualized by the indirect immunofluorescence technique. Each of the antisera produced specific and distinct patterns of fluorescence, suggesting that it is possible to use the indirect immunofluorescence technique to study the in situ organization of each histone in the various regions of the chromosomes. H1 and H2A antisera produced diffuse fluorescence patterns in acetic acid-fixed chromosomes which become more defined in formaldehyde-fixed preparations. Antisera to H2B, H3 and H4, when reacted with either formaldehyde- or acetic acid-fixed chromosomes, produce distinct banding patterns closely resembling the banding of acetoorcein-stained or phase-contrast-differentiated chromosomal preparations. These antisera produce corresponding patterns of fluorescence for each chromosome, suggesting that the overall organization of the histones is similar in the various bands. Because the dense band regions stain more brightly with antihistone sera than the less compacted interband areas, we believe that the number of antigenic sites of chromosome-bound histones is related to the amount of DNA present, and that the accessibility of histone determinants does not differ between the bands and interbands.     

Cerebral localization of insulin by immunofluorescence.

An immunohistochemical procedure was used to detect cells which appear to bind insulin in the mouse brain. Strong fluorescence was observed in the cell bodies and processes of tanycytes lining the third ventricle and in the choroid plexi. These findings suggest that insulin enters the central nervous system, and indicate a route for its possible transport. This adds credence to earlier observations that the hypothalamic ependymal cells and processes form a highly organized and functional system, with different cells selectively absorbing (or sensing) particular substances from the systemic and ventricular circulations and transporting them (or information about them) to specific neuron receptors in the hypothalamus.     

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.     

Angiotensin I-converting enzyme (ACE): estimation of DNA haplotypes in unrelated individuals using denaturing gradient gel blots

The angiotensin I-converting enzyme (ACE) gene (17q23) is a candidate gene for essential hypertension and related diseases, but investigation of its role in human pathology is hampered by a lack of identified polymorphisms. Currently, a 287-bp insertion/deletion (I/D) RFLP in intron 16 represents the only one known. Additional polymorphisms for the ACE gene would make most families informative for linkage studies and would allow haplotypes to be assigned in association studies. To increase the information provided by the ACE gene, we used a sensitive screening technique, denaturing gradient gel electrophoresis (DGGE) blots, to identify polymorphisms and combined this with gene counting to identify haplotypes. Five independent polymorphisms, restriction fragment melting polymorphisms (RFMPs), were identified by four probes (encompassing half of the ACE cDNA) in digests produced by three restriction enzymes (DdeI, RsaI, and AluI). One RFMP has three alleles while the others have two alleles. In a sample of 67 unrelated control subjects, minor allele frequencies ranged from 0.12 to 0.49. A significant level of linkage disequilibrium was found for all pairs of markers. The four most informative RFMPs, taken in combination, define 24 potential haplotypes. Based on gene counting, 11 of the 24 are rare or nonexistent in this population, and the estimated heterozygosity of the remaining 13 haplotypes approaches 80%. Under these conditions for the ACE locus, phase-unknown genotypes could be assigned to haplotype pairs in unrelated subjects with reasonable certainty. Thus, using DGGE blot technique for identifying numerous DNA polymorphisms in a candidate locus, in combination with gene counting, one can often identify DNA haplotypes for both related and unrelated study subjects at a candidate locus. These markers in the ACE gene should be useful for clinical and epidemiologic studies of the role of ACE in human disease.