T-Lymphocyte and Plasma Angiotensin-Converting Enzyme Activity During Enalapril and Losartan Administration in Humans

This study evaluated the long-term effects of the angiotensin-converting enzyme inhibitor enalapril and the angiotensin II type 1 receptor antagonist losartan on the angiotensin-converting enzyme activity in T lymphocytes and plasma in patients with essential hypertension. The study was a randomized, placebo-controlled, double-blind, crossover design. Nine patients with sitting blood pressure ≥ 95 mm Hg and ≤ 105 mm Hg at the end of a 4-week placebo run-in period entered the double-blind phase of the study, which consisted of three 6-week periods during which patients were treated with placebo, enalapril (20 mg, once daily), or losartan (50 mg, once daily) The angiotensin-converting enzyme activity in T lymphocytes was measured as the activity of the degradation of the substrate Hippuryl-His-Leu and as the appearance of the dipeptide His-Leu, which was quantified spectrofluorometrically. Enalapril but not losartan suppressed (p ≤ 0.01) the angiotensin-converting enzyme activity in plasma, whereas it stimulated (p ≤ 0.05) the angiotensin-converting enzyme activity in circulating T lymphocytes. Our data document induction of angiotensin-converting enzyme in human T lymphocytes during long-term treatment with the angiotensin-converting enzyme inhibitor enalapril. Angiotensin II receptor type 1 antagonism with losartan had no effect on plasma or lymphocytic angiotensin-converting enzyme.

Plasma angiotensin-converting enzyme (ACE) activity was found to be significantly decreased in patients with hypertension treated long term with ACE inhibitors, if it was measured during the lifetime of the inhibitor in plasma ^(1-3). However, long-term treatment with an ACE inhibitor increased the content and the activity of ACE in plasma of rats or patients ^(4-8) as well as in tissues of rats ^(9-12) when the inhibitor could be removed from the sample just before analysis.

The circulating blood cells are the most appropriate cells for studies in humans. If the effects of ACE inhibitors were identical in tissue and blood cells, then the latter could be used for the visualization of the induction of ACE in hypertrophied or fibrotic tissue after treatment with ACE inhibitors. The major blood cells expressing ACE are T lymphocytes ⁽¹³⁾.

The present study aimed to elucidate whether long-term treatment of patients with essential hypertension with the ACE inhibitor enalapril or with losartan, the first of a new class of antihypertensive agents that interfere with the renin angiotensin system by blocking the angiotensin II type 1 receptor, could affect ACE activity in T lymphocytes.

METHODS

Patients

The study protocol has been published in detail ⁽¹⁴⁾. White patients with essential hypertension, aged ≥ 18 years, in World Health Organization stages I and II, were eligible for the study. Patients with any disease that could interfere with the study protocol were excluded, as were women who were pregnant or receiving oral contraceptives. The protocol was approved by the Ethics Committee of the Faculty of Medicine of the University of Leuven, and participants gave written witnessed informed consent to participate in the study.

Treatment protocol

Eligible patients were invited for an initial visit after not receiving antihypertensive treatment for at least 2 weeks. They began a 4-week run-in period during which they received two placebo capsules each day. Patients were instructed to ingest the study medication in the morning with their breakfast, between 7 a.m. and 8 a.m., including on visit days; they were asked to keep general lifestyle, diet, and physical activity constant. Nine male patients satisfied the blood pressure criteria on the final day of placebo treatment (mean sitting diastolic blood pressure ≥ 95 mm Hg and ≤ 105 mm Hg) and were allocated to be administered placebo, the converting enzyme inhibitor enalapril (20 mg, once daily), or the angiotensin II receptor antagonist losartan (50 mg, once daily) in a randomized crossover study. The placebo period was always scheduled between the two active treatment periods and it was not known to the investigators who performed the measurements. Each treatment period lasted 6 weeks. Capsules containing placebo, enalapril, or losartan had the same shape and color.

Blood sampling

Blood was withdrawn from an antecubital vein into heparinized tubes for assay of routine biochemical variables, for determination of ACE activity in serum ⁽¹⁵⁾ and T lymphocytes. Blood was drawn between 8:30 a.m. and 10 a.m.

Isolation of peripheral blood mononuclear cells

Peripheral blood was diluted 1:2 with Hanks' balanced salt solution (GIBCO) and the peripheral blood mononuclear cells (PBMCs) were separated by centrifugation (560 g for 25 min) on Lymphoprep 1.077 (Nycomed Pharma AS, Oslo, Norway). The PBMC layer was harvested and washed thrice with Hanks' balanced salt solution, counted, and suspended in RPMI 1,640 medium (GIBCO BRL). This separation procedure was done at room temperature. The resulting preparation of mononuclear cells (n = 7) contained 88.2 ± 1.3% lymphocytes, 8.3 ± 1.2% monocytes, 1.59 ± 0.3% neutrophils, 0.28 ± 0.08% eosinophils, and 1.62 ± 0.35% basophils.

Cell purity determined by Wright staining was > 97% and cell viability exceeded 98% as assayed by the Trypan blue exclusion test.

Isolation of pure T lymphocytes

The obtained PBMCs (30.10 ⁶ /10 ml) were rolled at 4°C for 30 min and then placed upright in ice for 15 min. Approximately 7.5 ml was aspirated, transferred to a Falcon tube of 15 ml, and centrifuged at 300 g at -20°C for 10 min. The supernatant was aspirated and discarded and the precipitate was detached. Then 0.8 ml of Lympho-kwik LK-50T was added, and the precipitate transferred in an Eppendorf tube and rinsed again with 0.8 ml LK-50T. After mixing with a thermomixer (60 min at 37°C) and centrifugation (2,800 g for 2 min), the supernatant was aspirated, the precipitate detached, and 1 ml of RPMI added. After centrifugation (1,400 g for 1 min) the supernatant was aspirated and the precipitate detached.

The 100-μl monoclonal antibody to CD16 and CD56 was added together with 0.8 ml Lympho-kwik. After mixing with a thermomixer (60 min at 37°C) and centrifugation (2,800 g for 2 min), the supernatant was aspirated and the precipitate detached from the wall. One milliliter of RPMI was added and the tubes were centrifuged at 1,400 g for 1 min. The supernatant was aspirated, and the precipitate was detached and dissolved into the adequate medium. The obtained suspension contains only T-suppressor/cytotoxic cells.

These T cells were quantified by a fluorescence-activated cell sorter (FAC scan cytometer, Becton Dickinson, San José, CA, U.S.A.) using a Simul Test software package VI.I and counted routinely with a Sys Mex Microcellcounter F800 (Toa Medical Electronics, Kobe, Japan).

Homogenates of these T cells are prepared by alternate freezing in liquid nitrogen, thawing, and dissolving in Triton X-100. Therefore, the medium was aspirated and then 2.5 × 10 ⁶ cells dissolved in 100 μl of phosphate-buffered saline, pH 7.2, containing 9.3 m M Na ₂ HPO ₄ , 2.9 m M KH ₂ PO ₄ , 3 m M KCl, and 136 m M NaCl. Ten microliters of Triton X-100 (20%) was added. After alternate (three times) freezing, thawing, and centrifuging (10,000 g for 15 min) of this suspension, 10 μl of the supernatant was used freshly for the assay of ACE.

Assay of angiotensin-converting enzyme in homogenates of T lymphocytes

The fluorimetric assay for ACE in T lymphocytes is based on the conversion of the substrate analogue hippuryl-histidyl-leucine to hippurate and l -histidyl-leucine, which is quantified spectrofluorometrically (λ _excitation = 360 nm, λ _fluorescence = 500 nm) by formation of a fluorescent adduct with o-phthaldialdehyde ^(16,17).

Ten microliters of the T-lymphocyte homogenate were incubated with 5 μl of 250 m M hippuryl- l -histidyl- l -leucine in 50 μl of buffer pH 8.3 containing 0.5 M K ₂ HPO ₄ and 1.5 M NaCl plus 140 μl of distilled water for 18 h at 37°C. This reaction was stopped by the addition of 1.45 ml of 0.28 N NaOH. Then 100 μl of 2% o-phthaldialdehyde in methanol was added to each tube and mixed with a vortex. Exactly 10 min later this reaction was terminated by the addition of 200 μl of 3 N HCl and the tubes were centrifuged at 15,000 g for 10 min. The supernatant was transferred to a quartz fluorescence cuvette and the cuvette placed in a thermostated cuvette holder. The fluorescence was read, 30 min after the addition of HCl but within 90 min after its addition, with a LS-50B fluorometer (Perkin-Elmer, Norwalk, CT, U.S.A.). The excitation wavelength at a slit of 8 nm was 360 nm and the emission wavelength was 500 nm at a slit of 6 nm, with the use of a filter of 430 nm.

Statistical analysis

Database management and statistical analyses were performed using SAS software (SAS Institute Inc., Cary, NC, U.S.A.). Group data are reported as mean ± SD. Positively skewed data were transformed logarithmically for the statistical analyses. The effects of treatment were assessed by use of Wilcoxon signed rank test. Two-tailed p ≤ 0.05 was considered statistically significant.

RESULTS

General characteristics of the patients

Age of the nine men averaged 46 ± 6 years and body mass index was 26.9 ± 2.2 kg/m ² . Median duration of hypertension was 6 months (range, 1-120 months). Five patients had previously been treated for hypertension, and two were current smokers. Sitting blood pressure and heart rate at the end of the run-in period averaged 154 ± 13/102 ± 3 mm Hg and 75 ± 17 beats/min, respectively. Serum creatinine averaged 94 ± 12 μM and urinary sodium excretion 165 ± 67 mmol/24 h. All patients completed the double-blind protocol.

Angiotensin-converting enzyme activity

Plasma ACE activity was significantly decreased during enalapril treatment but not during losartan administration (Table 1). ACE activity in T-lymphocyte homogenates was not derived from fetal bovine serum because the medium taken after the last washing of the T lymphocytes did not show any activity of Hip-His-Leu degradation. The ACE activity in T lymphocytes was not changed after losartan treatment and increased (p < 0.05) with enalapril (Table 1). Figure 1 shows the ACE activity in plasma and T lymphocytes in the nine patients separately during enalapril, losartan, and placebo treatment.

Table 1

Fig. 1

DISCUSSION

In the current study, plasma ACE activity was decreased after long-term treatment of patients with hypertension with enalapril and was not affected by losartan treatment. The measurements of plasma ACE activity were performed during the lifetime of the ACE inhibitor (i.e., within 3 h after drug intake). Our data are in agreement with other studies in which plasma ACE activity was measured in similar conditions ^(1-3).

Despite the inhibited plasma ACE activity, long-term treatment with lisinopril induced an increase in plasma ACE content in rats ⁽⁶⁾. Long-term treatment of rats or patients with hypertension with ACE inhibitors also increased plasma ACE activity as compared with short-term treatment, if the inhibitor was removed from the sample before analysis ^{(4,5,8,11,18-21)}. This induction of ACE could be observed a long time after the last administration of the ACE inhibitor, exceeding the lifetime of the ACE inhibitor in plasma, as shown for captopril and lisinopril in rats ⁽⁸⁾ and in humans ⁽⁴⁾. An increase in the plasma ACE concentration has also been shown by the comparison of the dose-dependent effects of enalapril on the plasma ACE activity after short-and long-term treatment ^(19,20). Indeed after both short-and long-term treatment of patients with enalapril, the plasma concentration of the active metabolite enalaprilat was strongly related to the inhibition of the ACE activity, but the dose-dependence curve after long-term treatment was shifted to the right as compared with the curve after short-term treatment ^(19,20). The IC ₅₀ after long-term enalapril administration was two times greater than its value after short-term administration. This means that long-term administration of enalapril in humans was associated with an induction of the enzyme in plasma ^(19,20).

The increase in the ACE concentration in plasma and tissue after long-term treatment with ACE inhibitors could result from a decrease in clearance of the inhibitor-enzyme complex, an increase in the release of this complex from the cell membrane, or from an increased synthesis and secretion of ACE by cells ⁽⁵⁾. Indeed it has been shown that captopril induces an increase in the secretion of ACE by cultured endothelial cells ^(4,22).

In human studies, blood cells containing ACE are appropriate cells for the investigation of the effects of ACE inhibitors on cell ACE activity. T lymphocytes are the major cell type expressing ACE in blood ^(13,23,24). Enalaprilat inhibited ACE in human T lymphocytes in vitro, when it was present in cell suspensions or homogenates ⁽¹³⁾. However, as shown in the current study, treatment of patients with enalapril increases ACE activity in lymphocytes in vivo.

If the ACE inhibitor were bound irreversibly by the enzyme, then the ACE activity in lymphocytes could be inhibited even after washing of the cells. However, we did not find any inhibition in the current study (Table 1). Conversely, if an ACE inhibitor dissociates completely from ACE during cell washing, then the measured ACE activity is a true value characterizing lymphocytes in the absence of an inhibitor. In this case, if long-term treatment with an ACE inhibitor increases the ACE activity in lymphocytes (either directly or via the induction of ACE synthesis), then after purification of cells from the blood the ACE activity per cell could be higher as compared with placebo. Indeed we found that ACE in circulating T lymphocytes was increased after treatment of hypertensive patients with enalapril. It has previously been demonstrated that long-term treatment with captopril increases the content of ACE in tissues such as rat lung ^(5,10,11,22); kidney ^(5,11); and heart and aorta ⁽⁵⁾. Treatment of dogs with congestive heart failure induced angiotensin II formation in the heart ⁽²⁵⁾. In rats, lisinopril increased ACE in lung ⁽⁶⁾ and quinapril increased ACE mRNA in left and right ventricles ⁽¹²⁾. Costerousse et al. ⁽²⁶⁾ also compared the effect of ACE inhibition, angiotensin II antagonism, and renin inhibition on ACE gene expression and tissue level in the rat. Their results showed that ACE gene expression and enzyme concentration during ACE inhibition are increased in tissues such as heart, lung, and duodenum. In vivo administration of ACE inhibitors thus induced ACE expression in cells in tissues. In the current study we have shown that in vivo treatment with the ACE inhibitor enalapril also increases the ACE activity in circulating T lymphocytes.

In conclusion, our data show two potentially interesting observations: the first demonstration at the cellular level in humans of induction of ACE synthesis during long-term ACE inhibition; and further evidence against a retrocontrol of ACE synthesis by angiotensin II, as was proposed by some investigators ⁽⁸⁾.

Acknowledgment:

The authors gratefully acknowledge the technical and secretarial assistance of Miss T. Coenen, Miss L. Lommelen, Miss Y. Piccart, and Mrs Y. Toremans. This work was supported by the Medical Grant Program from Merck, Sharp & Dohme (U.S.A.). R. Fagard is holder of the A. Amery Chair in Hypertension Research founded by Merck, Sharp & Dohme, Belgium. P. Lijnen is holder of the Boehringer Ingelheim Chair in Hypertension.