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(Hypertension. 1995;26:1145-1148.)
© 1995 American Heart Association, Inc.

Articles

Calcium Channel Blockers as Inhibitors of Angiotensin I–Converting Enzyme

Dulce E. Casarini; Adriana K. Carmona; Frida L. Plavnik; Maria T. Zanella; Luiz Juliano; Artur B. Ribeiro

From the Department of Medicine, Divisions of Nephrology (D.E.C., F.L.P., A.B.R.) and Endocrinology (M.T.Z.), and Department of Biophysics (A.K.C., L.J.), Escola Paulista de Medicina, Universidade Federal de São Paulo (Brazil).

Correspondence to Dulce E. Casarini, Department of Medicine, Division of Nephrology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu 740, 04023-062, São Paulo, SP, Brazil.

	Abstract

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Abstract Using ion-exchange chromatography of dialyzed human urine from healthy and hypertensive patients, we detected two peaks of angiotensin I–converting enzyme (ACE) activity on hippuryl-His-Leu eluted at ionic strengths of 0.7 (F1 peak) and 1.25 (F2 peak) mS. These hydrolytic activities decreased gradually in the urine of patients submitted to isradipine treatment, F2 and F1 disappearing after 12 and 24 hours, respectively. By Western blot analysis, the urine fractions corresponding to both peaks from healthy and untreated patients presenting ACE activity and from treated patients (24 hours) without this activity were recognized by an ACE-specific antibody. These results indicated that ACE was present but inhibited in the urine of isradipine-treated patients. In vitro assays with ACE isolated from human urine and guinea pig plasma demonstrated that the enzyme is inhibited by isradipine and other commercially available calcium channel blockers, such as felodipine, nifedipine, and verapamil. A noncompetitive inhibition was observed with all calcium channel blockers studied. In conclusion, these results suggest that besides the primary effect on calcium channels, the more commonly used calcium channel blockers are also ACE inhibitors. The development of efficient calcium channel blockers with higher ACE inhibitory activity could result in interesting bifunctional antihypertensive drugs.

Key Words: angiotensin-converting enzyme • calcium channel blockers • isradipine

	Introduction

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Angiotensin-converting enzyme (ACE) (EC 3.4.15.1), a carboxy-dipeptidase that plays an important role in blood pressure regulation,¹ cleaves the C-terminal dipeptide from angiotensin I, producing the potent vasopressor peptide angiotensin II,² and inactivates bradykinin, a potent vasodilator, by two sequential dipeptide hydrolytic cleavages.³ ACE is found in most mammalian tissues and is bound to the external surface of endothelial, epithelial, neural, and neuroepithelial membranes. This enzyme can be fully solubilized from the membranes by proteolytic cleavage,⁴ which is the form detected in plasma and other body fluids.⁵

Two ACE isoforms have been described: a somatic form in the endothelium and a germinal form in testis. The somatic ACE is a glycoprotein composed of a single polypeptide chain (molecular weight, 170 kD) containing two large homologous domains, each bearing an active catalytic site.⁶ The germinal form of ACE has a lower molecular weight (110 kD) and contains only one active site, which corresponds to the C-terminal domain.⁷

ACE of molecular weights ranging from 100 to 400 kD has been isolated from human urine of healthy individuals.^{8 9} We have previously detected by ion-exchange chromatography of urine from normotensive subjects two peaks of ACE activity with molecular weights of 170 and 60 kD (D.E.C. et al, unpublished data, 1995). A similar chromatographic profile, with peaks of molecular weights of 90 and 60 kD, was also found in hypertensive patients¹⁰ ; after 4 weeks of treatment with chlorthalidone the ACE of higher molecular weight persisted, whereas the other one disappeared.¹¹

In this article we report the disappearance of these two ACE forms in chromatographed urine from hypertensive patients treated with isradipine, a calcium channel blocker (CCB). In addition, we studied the direct effect of this drug and other usual CCBs, such as felodipine, nifedipine, and verapamil, as inhibitors of ACE isolated from guinea pig plasma and urine of healthy individuals.

	Methods

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Cellex D resin (DEAE-cellulose) and Bio-Gel A_0.5m were purchased from Bio-Rad. Sephadex G-200, Superose-12, and Mono Q were from Pharmacia LKB Biotechnology. Hip-His-Leu and His-Leu were synthesized by a classic solution method and characterized by analytical high-performance liquid chromatography and amino acid analysis.¹² Enalapril was supplied by Merck Sharp & Dohme, captopril by the Squibb Institute for Medical Research, nifedipine by Bayer, verapamil by Knoll, isradipine by Sandoz S/A, and felodipine by Merrell Lepetit LTDA. o-Phthaldialdehyde was purchased from Sigma Chemical Co. Polyvinylidine difluoride microporous membrane was purchased from Millipore. Antiserum Y4 and wild-type recombinant ACE were generous gifts from Dr François Alhenc-Gelas, Paris, France. All other chemicals were reagent grade or equivalent.

The human subjects studied were the same as those previously reported.¹³ The protocol was approved by the Ethics Committee on Human Experimentation (Hospital São Paulo, Universidade Federal de São Paulo). Ten hypertensive patients were admitted to the emergency unit of Hospital São Paulo. All patients were symptomatic at the moment of admission, but after further evaluation none of them presented any evidence of end-organ damage as seen on funduscopic examination, serum urea, creatinine, electrolyte levels, and urinalysis. Blood pressure levels were greater than 200 and 110 mm Hg for systolic and diastolic pressures, respectively.

All patients agreed to participate in the study and gave written informed consent. The study was conducted in two phases. In the first, patients were confined to bed, and during 1 hour normal saline was infused with a Harvard pump. In the second phase the patients received infusions of 1.2, 2.4, 4.8, and 7.2 µg/kg per hour isradipine during 3 hours for each drug concentration. Immediately at the end of these 12 hours of infusion, the patients received 2.5 or 5.0 mg isradipine orally.

Blood pressure was recorded at regular 10-minute intervals during the infusion period by an automated blood pressure recording device. Urine samples were collected after 1 hour of saline infusion (basal), at each infusion level during 24 hours, and at the first oral dose administration.

For partial purification of human urine from hypertensive patients by ion-exchange chromatography, urine samples collected during the infusion period were dialyzed for 24 hours against 0.02 mol/L sodium phosphate buffer, pH 7.0. Aliquots containing approximately 80 mg protein were applied to a DEAE-cellulose column (4.3x1.6 cm) equilibrated with 0.02 mol/L sodium phosphate buffer, pH 7.0, followed by an NaCl gradient from 0.02 to 0.50 mol/L in starting buffer.

Human urinary ACE from healthy individuals was purified by ion-exchange chromatography in a DEAE-cellulose column, followed by gel filtration on a Bio-Gel A_0.5m, as previously described.¹⁴ Plasmatic guinea pig ACE was purified from ammonium sulfate precipitate (1.4 to 2.8 mol/L) of guinea pig plasma followed by dialysis, gel filtration on Sephadex G-200 and Superose-12, and ion-exchange chromatography in a Mono Q column (A.K.C., L.J., unpublished data, 1995). Protein elution profiles were monitored by absorbance at 280 nm, and protein concentration was determined by Spector's method,¹⁵ using bovine serum albumin as standard.

Enzymatic activity was measured by the method of Cushman and Cheung¹⁶ adapted to a fluorometric procedure as described by Friedland and Silverstein.¹⁷ The assays using Hip-His-Leu were carried out in 0.1 mol/L potassium phosphate buffer, pH 8.3, containing 0.3 mol/L NaCl at 37°C; His-Leu release was quantified fluorometrically by the formation of a fluorescent adduct with o-phthaldialdehyde. K_m values were calculated by the Lineweaver-Burk plots of initial hydrolysis velocities for five substrate concentrations. Inhibition constants (K_i) were determined under the same conditions after a 30-minute preincubation of the inhibitors with ACE. Kinetic parameters were calculated with the GRAFIT computer program.¹⁸

The urine of treated and untreated patients was chromatographed in the same conditions, and the fractions corresponding to conductances at 0.7 and 1.25 mS were analyzed simultaneously with the ACE isolated from the urine of healthy individuals¹⁴ by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in a 7.5% polyacrylamide gel,¹⁹ followed by the Western blot procedure. After electrophoresis, proteins were transferred to a polyvinylidine difluoride microporous membrane and incubated overnight at 4°C with antiserum Y4 (1:1000). Subsequent steps were carried out by the usual development procedure with the streptavidin and phosphatase alkaline system.

	Results

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Isradipine infusion resulted in decreases in both systolic and diastolic values. Mean basal arterial pressure decreased progressively after each infusion from 135.2±4.4 mm Hg to 130.4±4.6, 128.6±4.0, 124.4±3.4, and 116.2±3.6 mm Hg at sequential rates of infusion, as previously described.¹³

Two peaks (F1 and F2) with ACE activity on Hip-His-Leu were eluted by ion-exchange chromatography of urine from untreated patients (Fig 1A). The ACE activity corresponding to peak F1, which eluted at 0.7±0.21 mS (n=9), had a molecular weight of 90 kD, and the ACE activity corresponding to peak F2, which eluted at 1.25±0.19 mS (n=8), had a molecular weight of 60 kD, as previously determined by SDS-PAGE.¹¹ The urine of hypertensive patients submitted to 10 hours of isradipine infusion when chromatographed in DEAE-cellulose showed that ACE activity corresponding to peak F2 disappeared, whereas that corresponding to F1 decreased (Fig 1B). After 24 hours of treatment, ACE activity corresponding to peaks F1 and F2 could not be detected (Fig 1C).

View larger version (17K): [in this window] [in a new window]   Figure 1. Plots show chromatography on DEAE-cellulose columns in basal conditions (A) and after 10 (B) and 24 (C) hours of isradipine infusion, as described in "Methods." indicates angiotensin-converting enzyme activity; , conductivity.

Human urinary ACE purified from healthy subjects and hypertensive patients before and after isradipine treatment was analyzed by SDS-PAGE and studied by Western blot with Y4 specific antibody (Fig 2). The antiserum Y4 recognized all urinary human ACEs from healthy subjects (lanes 1 and 2) and untreated patients (lanes 3 and 4). Lanes 5 and 6 were spotted with the fractions that correspond to ACE activities (peaks F1 and F2) shown by chromatography of the urine from patients treated with isradipine during 24 hours. Although no ACE activity toward Hip-His-Leu was detected in these fractions, the immunoblotting showed that the enzymes were present.

View larger version (37K): [in this window] [in a new window]   Figure 2. Western blot shows analysis of purified angiotensin-converting enzyme (ACE) in healthy subjects (peak F1: lane 1, MW=170 kD; peak F2: lane 2, MW=60 kD), untreated patients (basal; F1: lane 3, MW=90 kD; F2: lane 4, MW=60 kD), and treated patients after 24 hours of isradipine infusion (F1: lane 5, MW=90 kD; F2: lane 6, MW=60 kD). Lane 7 shows wild-type recombinant ACE. Samples (10 µg protein) were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western blotting with the antiserum Y4 raised against human kidney ACE. Arrows indicate positions of molecular mass markers (kD).

The K_m values determined for hydrolysis of Hip-His-Leu by ACE from guinea pig plasma (5.8x10^-3 mol/L) and human urine (2.3x10^-3 mol/L) were similar to those previously described for ACE from the same sources.^{8 20} Guinea pig plasma and human urinary ACE were noncompetitively inhibited by all the CCBs tested, in contrast to a competitive inhibition observed with captopril and enalapril. Fig 3 shows the inhibition curves obtained with isradipine and enalapril for both enzymes; the Table presents the inhibition constants (K_i) for captopril, enalapril, and all the CCBs studied.

View larger version (13K): [in this window] [in a new window]   Figure 3. Line graphs show effects of various concentrations of Hip-His-Leu on activity of angiotensin-converting enzyme (ACE) from guinea pig plasma and urine of healthy patients. A, Lineweaver-Burk representation for guinea pig plasma ACE in the absence () and presence of 2.5x10-6 mol/L enalapril () and 4.8x10-5 mol/L isradipine (). B, Lineweaver-Burk representation for human urine ACE in the absence () and presence of 2.5x10-6 mol/L enalapril () and 1.0x10-6 mol/L isradipine ().

View this table: [in this window] [in a new window]   Table 1. Inhibition of Guinea Pig Plasma and Human Urine Angiotensin I–Converting Enzyme

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study we demonstrate the disappearance of ACE activity in the urine of hypertensive patients after 24 hours of isradipine treatment. However, we detected in the ion-exchange chromatographic fractions of this urine the presence of immunoreactive material, recognized as ACE by a specific antibody, that corresponded to equivalent fractions from untreated patients, detected with similar ion-exchange chromatography of urine samples, that presented both ACE activity and immunoreactivity. In contrast to competitive inhibition by captopril and enalapril of ACE isolated from guinea pig plasma and from urine of healthy individuals, we observed noncompetitive inhibitory effects of isradipine and other usual CCBs, such as felodipine, nifedipine, and verapamil, on these enzymes. These results indicate that the disappearance of ACE activity in the urine of isradipine-treated patients might be due to inhibition of the two ACE forms. Although the K_i values for ACE inhibition by the CCB studied are quite high compared with those obtained for captopril and enalapril, the complete disappearance of ACE activity in the urine of patients after 24 hours of isradipine treatment could be related to a possible concentration of this compound in urine.

The noncompetitive ACE inhibition by CCBs indicates that these compounds do not interact with the active site of the enzyme. This could be due to the very dissimilar chemical structures of the CCB and ACE substrates or competitive inhibitors.

Pharmacokinetic studies with isradipine have shown that after 3 hours of oral administration of 5 to 20 mg of the drug, concentrations of 2 to 10 µg/L are found in the circulation, and 70% of this amount is excreted in urine.²¹ In our study we administered 2.5 to 5 mg isradipine orally, and these concentrations might be sufficient to inhibit urinary ACE activity in a 24-hour period. This suggests that the usual dose of oral isradipine used for treatment of hypertension may have an ACE inhibitory effect, although the clinical significance of this finding cannot be explained yet, and further studies are necessary to clarify this mechanism.

On the other hand, we also cannot rule out the possibility that the blood pressure fall contributed to ACE inhibition because similar results were found when ß-blockers and diuretics were used in hypertensive therapy.²²

In conclusion, the results presented in this report could be the starting point for the design of compounds with bifunctional antihypertensive effects.

Received June 18, 1995; first decision September 19, 1995; accepted October 10, 1995.

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