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Affinity of Angiotensin I-Converting Enzyme (ACE) Inhibitors For N- and C-Binding Sites of Human ACE Is Different in Heart, Lung, Arteries, and Veins

Bevilacqua, Maurizio; Vago, Tarcisio; Rogolino, Angela; Conci, Fabrizio; Santoli, Edoardo; Norbiato, Guido

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Journal of Cardiovascular Pharmacology: October 1996 - Volume 28 - Issue 4 - p 494-499
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Abstract

Angiotensin-converting enzyme (ACE: E.C. 3.4.15.1), a carboxy peptidase, is present in different concentrations in various animal and human organs (1-3). The enzyme is characterized by two homologous domains, both of which are catalytically active (4-11), a C-domain that cleaves angiotensin-I and bradykinin, and an N-domain that may preferentially cleave luteinizing hormone-releasing hormone (12,13) and seraspenide, a protein that has an important regulatory role in myelopoiesis (14). The involvement of ACE in the regulation of seraspenide levels was also recently demonstrated in in vivo studies of humans by Corvol and associates (15), who showed that the administration of captopril, an N-seeking ACE inhibitor (ACEI) followed by a long-lasting 5.5-fold increase in the plasma levels of seraspenide, suggesting that ACE inhibition could have hematological implications and also play an important immunological role. Indeed, T lymphocytes (16) and monocytes (17), i.e., the cells that present the antigen in some immune reactions, are characterized by the presence of constitutive and/or inducible mRNA of ACE. The stimulation of the immune response by class I-restricted antigenic peptides, such as the human immunodeficiency virus-1 (HIV-1), glycoprotein (gp) 160-derived peptide, requires peptide processing by ACE (18). Therefore, ACEI may affect the presentation of this antigen to T lymphocytes, thus modulating the immune response (18,19). Another feature of ACE inhibition is the ability of captopril to counteract lung fibrosis in irradiated rats (20), possibly through the inhibition of fibroblast proliferation (21), and to contrast duodenal injury induced by irradiation (22).

ACE exists in various biochemical forms, i.e., with both catalytic sites (C- and N-terminals) (1-3), with N-terminal site only (ileal fluid ACE) (23), with C- and N-terminal sites, but not with the peptide that enchains ACE to the plasma membrane (circulating ACE) (24,25) and with the C-site only (testis) (3,26-29). According to the model of Perich and colleagues (30), ACEI, with increased hydrophobicity and a voluminous side chain bind C-site with greater affinity than N-site. By contrast, ACEI with a small side chain and nonhydrophobic properties bind preferentially to N-site. In a further development of their hypothesis, Perich and colleagues (31) reported that the ability of captopril, an ACEI with a small moiety and low hydrophobicity, to recognize the N-site better is due to its own sulfhydrilic nature, whereas carboxyalkyl derivatives with a big side chain recognize with C-site greater affinity. Phosphorous-containing ACEI recognizes both sites with the same affinity. These findings were obtained in rat tissues, which show some differences in ACE number and some affinity with respect to human beings (32). We performed some experiments to evaluate the number and the affinities of C- and N-sites in various human organs. As a radioligand, we used [125I]Ro 31-8472, a cilazaprilat derivative that does not discriminate between the N- and C-site of ACE (8). As ACEI, we used captopril because of its high affinity for N-site and delaprilat, the active metabolite of delapril, an ACEI characterized by a very large side chain and by increased hydrophobicity. N- and C-selectivity of these drugs was validated by comparison between their binding characteristics on rat testis and lung tissue (30). Fig. 1

METHODS

Source of tissues

We obtained 10-20 g left ventricular (LV) anterior wall and coronary arteries from 3 prospective organ donors and 3 industrial accident victims (5 men, 1 woman; mean age 35 ± 4 years). After being declared unsuitable for transplantation, their hearts were obtained at autopsy within 3 h of death. No evidence of medication was noted in the medical histories of the donors. Human lung was obtained from the same source. Mammary arteries were obtained as previously reported (33). Human saphenous veins were obtained from the cardiovascular surgery department from patients undergoing saphenectomy for coronary aortic bypass procedures. Rat testis and lung were taken from Wistar Kyoto males (32).

Plasma membrane preparation

The tissues were freed from connective tissue, fat was trimmed with scissors, and tissue was pulverized under liquid nitrogen with a tissue dismembrator (Mikro-Dismembrator II, Braun, Melsungen, Germany). The pulverized tissues were resuspended in buffer A (composition: Tris-HCl 20 mM, NaCl 125 mM, KCl 10 mM, sodium acetate 10 M, D-glucose 5 mM, zinc sulfate 50 μM, pH 7.4 at 20°C). In some experiments, we evaluated the zinc dependency of binding by preparing the membranes in the same buffer without zinc sulfate (buffer B) or in the presence of EDTA (buffer C). Preliminary experiments showed that the binding of [125I]Ro 31-8472 to plasma membranes was absent in the presence of EDTA and that it was highly variable in the absence of zinc sulfate; therefore, in the experiments, as regular procedure, we used buffer A. The pulverized tissues were resuspended in buffers and centrifuged at 600 g for 10 min at 4°C. The supernatant was recovered and centrifuged at 48,000 g for 35 min at 4°C. The pellet was resuspended in buffer A and either assayed immediately for [125I]Ro 31-8472 binding or stored in liquid nitrogen.

Preparation of [125I]Ro31-8472

Ro31-8472, [9-[1-carboxy-3-(4-hydroxyphenyl 1) propylamino]-octahydro-10-oxo-6H-pyridazo [1,2-9][1,2] diazepine] 1-carboxylic acid] was synthetized by Roche (Hertfordshire, U.K.) and donated by Dr. M. R. Attwood. Ro31-8472 was iodinated by Hunter's and Greenwood's chloramine-T method (34) under conditions previously described by Perich and colleagues (8).

Binding assay

The rat testis (2 μg/tube), rat lung (2 μg/tube), and the human cardiac (10 μg/tube), lung (2 μg/tube), artery (10 μg/tube), and vein (10 μg/tube) homogenates were incubated for 5 h at room temperature (20°C) in siloxanized glass tubes containing 0.025-1.5 nM [125I]Ro 31-8472 in buffer A in a 400-μl vol. Non specific binding was determined in the presence of 1 μM enalaprilat. We stopped the reactions with the addition of 3 ml ice-cold buffer A and rapid filtration of samples through Whatman GF/C filters (Whatman International, Maidstone, U.K.). Filters were then quickly rinsed with 2 × 10 ml buffer A. The filter-bound radioactivity was measured in a γ-counter. Competition experiments were performed in the same manner but with 0.1 nM[125I]Ro30-8472 and various concentrations of competing ligands from 10-11 to 10-4M. After incubation, we evaluated the radioactivity bound to plasma membranes with a γ-counter, as previously described (35).

Protein measurement

We measured the protein content of plasma membranes with the assay described by Bradford (36). Fig. 2

Drugs and chemicals

We tested the following ACEI: enalaprilat, the active metabolite of enalapril (Merck Sharp and Dohme, Rahway, NJ, U.S.A.); captopril (Squibb, Paris la Défense, France); and delaprilat, the active metabolite of delapril (Chiesi, Parma, Italy) (31); and Ro31-8472 (Roche). Other chemicals were analyticalgrade reagents obtained from usual commercial sources. Fig. 3

Data analysis

The saturation binding isotherms of [125I]Ro31-8472 to ACE binding sites and the competition isotherms of the same ligand in the presence of increasing concentrations of competing ACEI were analyzed by INplot 4 (Graph-Pad, San Diego, CA, U.S.A.) (37). The data were expressed as Hill number (nH) (38) and as pKi (pKi = -log Ki), where Ki is the binding affinity constant of unlabeled ligand.

Statistical analysis

Binding data were expressed as the mean ± SD of n experiments. We compared the pKi values by analysis of variance (ANOVA).

RESULTS

Validation of captopril and delaprilat as N- and C-site selective or nonselective pharmacological probes

Saturation experiments with [125I]Ro 31-8472 in rat lung and testis membrane preparation were monophasic and disclosed ACE sites of 13,817 ± 1,140 fmol/mg (Kd = 0.08 ± 0.01 pM) in testis and of 20,141 ± 3,515 fmol/mg (Kd = 0.10 ± 0.02 pM) in lung. Fig. 4

Competition isotherms with captopril were characterized by an affinity for the C-sites (testis) (pKi = 7.00 ± 0.10), which is very similar to the lower affinity site of the rat lung (pKi = 6.86 ± 0.09). Delaprilat displayed a high affinity for testis ACE (pKi = 9.90 ± 0.08) comparable to the higher affinity site of the rat lung (pKi = 9.83 ± 0.07). Enalaprilat and Ro 31-8472 displayed a single site affinity, both on testis and lung, of the same affinity order (Table 1). Fig. 5

The binding of [125I]Ro 31-8472 to human heart LV homogenate was saturable and exhibited a binding site density of 560 ± 65 fmol/mg protein with a Kd of 0.36 ± 0.05 pM (n = 5). Human lung displayed 17,680 ± 2,345 fmol/mg protein of ACE binding sites with a Kd = 0.32 ± 0.04 fmol/mg protein. Human coronaries displayed 237 ± 51 fmol/mg protein of ACE binding sites with a Kd = 0.37 ± 0.06 nM. Human mammary artery displayed 603 ± 121 fmol/mg protein of ACE binding sites with a Kd = 0.50 ± 0.06 nM. Human saphenous veins displayed 236 ± 63 fmol/mg protein with a Kd = 0.14 ± 0.05 (n = 4). The relative affinities of captopril and delaprilat for N- and C-site in human tissues are shown in Table 2.

The pKi values of captopril on the N-site of lung and mammary artery were significantly higher than pKi values on the N-site in myocardium, coronary artery, and saphenous vein (p < 0.01, ANOVA). The pKi values of delaprilat on the C-site in lung and mammary artery were less than pKi values on the C-site in myocardium and coronary artery (p < 0.01, ANOVA).

DISCUSSION

We used a rat testis/lung system (30) to characterize some ACEI in light of their affinity for N- and C-site of ACE. With this aim, we evaluated the ACEI on rat testis ACE, which has only the carboxy terminal half of somatic ACE (3). We showed that captopril was characterized by a low affinity for this site. Such affinity was similar to the low affinity site of the two active sites of lung membranes. On the contrary, delaprilat showed high affinity for the C-site of rat testis, an affinity very similar to that of delaprilat on the high-affinity site in the rat lung. In accordance with their affinities, these two drugs were selected as C-selective (delaprilat) and N-selective (captopril). Enalaprilat did not show biphasism and was chosen as a nonselective drug. Ro 31-8472, used as ligand, was monophasic and therefore suitable for labeling the sites. This ligand does not have the site affinity of other ligands (39,40), and thus enables a thorough investigation of the characteristics of the N- and C-sites in rat tissues.

In recent years, two models have been proposed to explain the different binding characteristics of various ACEI on N- and C-sites. Perich and colleagues (30) proposed that a decrease in the affinity for the N-site was directly related to an increased side chain and/or increased hydrophobicity of the ACE inhibitors tested. The trend was reversed at the C-site, where drugs with a big side chain and increased hydrophobicity showed a rather high affinity. This model fits well with our data being delaprilat, which disclosed a higher affinity for the C-site, characterized by a side chain (41) longer than that of ACEI currently used in humans and also highly hydrophobic. Recently, the same group of researchers (31) proposed that sulfhydril-containing ACEI are N-site selective, that carboxyalkyl derivatives are C-site selective, and that phosphinyl derivatives are equally active on both sites. This model does not fit with our data since enalaprilat and Ro 31-8472, two carboxyalkyl derivatives, were clearly monophasic in all tissues tested. Fig. 6

In human somatic tissues, captopril, delaprilat, and enalaprilat maintained the characteristics of phasism demonstrated in rat lung, but showed different affinities. The main finding of the present study is that in different human organs involved in the cardiovascular activity of ACEI (42-46), the affinity of an N-seeking drug (captopril) may vary 10-fold and that of a C-seeking drug (delaprilat) may vary ≈7-fold. In particular, captopril disclosed a relatively high affinity for the N-site of ACE in lung and mammary artery, whereas delaprilat showed an increased affinity for the C-site of the heart left ventricle and of the coronary artery. The reason for the different affinities of C- and N-sites in various tissues is unknown. The gene that encodes ACE is the same in all tissues, and the expression of the C-site only in testicular tissue is linked to a postsplicing phenomenon (26). However, ACE is heavily glycosylated and glycosyl residues may confer a different access to the enzymatic sites (47-49). Posttranslational glycosylation is organ dependent, and, in some experiments, the changes in ACE glycosylation conferred different biochemical properties (50). Fig. 7

That among the tissues tested, the lung and mammary arteries appeared to display a high affinity for captopril (N-site) and a relatively low affinity for delaprilat (C-site) is noteworthy. The opposite was true of myocardium and coronary arteries, which, on the whole, exhibited low affinity for captopril (N-site) and a relatively high affinity for delaprilat (C-site). The smooth muscle cells of the abdominal region derive from locally recruited mesenchymal smooth muscle, whereas the smooth muscle cells of the thoracic region derive from the neural crest (ectomesenchymal smooth muscle). Topouzis and co-workers (51) reported that coincubation of ectomesenchymal smooth muscle with endothelial cells prompted a larger increase in endothelial ACE activity with respect to the coincubation with mesenchymal smooth muscle. Furthermore, ACE activity was higher in thoracic than in abdominal vessels and was controlled by the neural crest, in that neural crest ablation modified thoracic but not abdominal ACE activity. Neural crest (52) is the precursor of ectomesenchymal organs, i.e., the cusps of arterial valves (extraordinarily rich in ACE activity), the tunica media of the large arteries derived from the aortic arch arteries (characterized by levels of ACE activity higher than those derived from mesenchymal organs) (51), the thymus (noteworthy are the high levels of ACE mRNA in T lymphocytes and the thymic origin of seraspenide, a polypeptide cleaved by N terminal of ACE), the parathyroid, and the thyroid glands. In a previous article (35), we hypothesized that the organs with a neuroectodermal component (lung, mammary artery, and atrium) may have different numbers and properties of ACE from organs with mesenchymal origin, such as coronary arteries, saphenous vein, and heart (53). A posttranslational mechanism may affect glycosylation on an ontogenetic basis (54), thus conferring increased ACE activity and a higher N-selectivity in organ with ectomesenchymal components with respect to organ of mere mesenchymal origin. Fig. 8

FIG. 1.
FIG. 1.:
Competition isotherms of captopril on rat testis (characterized by the C-site only) and lung plasma membranes.
FIG. 2.
FIG. 2.:
Competition isotherms of delaprilat on rat testis (characterized by the C-site only) and lung plasma membranes.
FIG. 3.
FIG. 3.:
Competition isotherms of enalaprilat on rat testis (characterized by the C-site only) and lung plasma membranes.
FIG. 4.
FIG. 4.:
Competition isotherms of captopril, delaprilat, and enalaprilat on human left ventricle plasma membranes.
FIG. 5.
FIG. 5.:
Competition isotherms of captopril, delaprilat, and enalaprilat on human lung plasma membranes.
FIG. 6.
FIG. 6.:
Competition isotherms of captopril, delaprilat, and enalaprilat on human saphenous vein plasma membranes.
FIG. 7.
FIG. 7.:
Competition isotherms of captopril, delaprilat, and enalaprilat on human mammary artery plasma membranes.
FIG. 8.
FIG. 8.:
Competition isotherms of captopril, delaprilat, and enalaprilat on human coronary plasma membranes.

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Keywords:

Angiotensin-converting enzyme; N Site; C Site; Captopril; Delapril

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