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Cardiovascular Research 1998 40(1):206-210; doi:10.1016/S0008-6363(98)00103-5
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Upregulation of angiotensin converting enzyme by atrial natriuretic peptide and cyclic GMP in human endothelial cells

Outi Saijonmaa* and Frej Fyhrquist

Minerva Institute for Medical Research and Department of Internal Medicine, Helsinki University Central Hospital, Helsinki, Finland

* Corresponding author. Address for correspondence: Minerva Institute for Medical Research, Tukholmankatu 2, SF-00250 Helsinki, Finland. Tel.: +358 (0) 477 1004; Fax: +358 (0) 477 1025; E-mail: outi.saijonmaa@kruuna.helsinki.fi

Received 12 November 1997; accepted 6 March 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: To examine the role of atrial natriuretic peptide (ANP) and cyclic GMP in the regulation of angiotensin converting enzyme (ACE) in cultured human endothelial cells. Methods: Cultured endothelial cells from human umbilical veins (HUVEC) were treated with ANP (0.3–30 nM), 8-Br-cGMP (1–100 µM), Rp-8-Br-PET-cGMPS (1 µM), or the phosphodiesterase inhibitors, zaprinast (10–100 µM), dipyridamole (1–10 µM), or isobutyl methyl xanthine (IBMX, 0.1–0.5 mM). ACE amounts were measured by inhibitor binding assay and cellular cGMP levels by radioimmunoassay. Results: ANP caused a dose dependent increase in ACE measured in intact endothelial cell culture. The stimulatory effect of ANP was blocked by Rp-8-Br-PET-cGMPS, a protein kinase G inhibitor. The cyclic GMP analog, 8-Br-cGMP and the cyclic GMP specific phosphodiesterase inhibitor, zaprinast, both increased ACE. Increase of ACE was also caused by nonspecific phosphodiesterase inhibitors, dipyridamole and IBMX. Intracellular cGMP levels were shown to increase by ANP, and phosphodiesterase inhibitors. Conclusions: These data suggest that cGMP is an intracellular mediator regulating ACE and that ANP induced increase of ACE is mediated via a cGMP dependent mechanism.

KEYWORDS Angiotensin converting enzyme; Atrial natriuretic peptide; Cyclic GMP; Phosphodiesterase inhibitors; HUVEC


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Angiotensin converting enzyme (ACE) is a dipeptidyl carboxypeptidase widely distributed on the luminal surface of vascular endothelium. ACE catalyzes the proteolytic cleavage of angiotensin I to angiotensin II (AII) and has bradykinin degrading activity [1]. Thus, ACE participates in the control of vascular resistance by generating AII and degrading bradykinin. AII also acts as a vascular growth factor participating in angiogenesis, vascular remodelling and response to vascular wall injury [2]. As a regulator of AII production ACE may have an important role in cardiovascular diseases such as atherosclerosis and hypertension. Thus, ACE inhibitors are effective both in reducing experimental atherogenesis [3]and in the reduction of left ventricular hypertrophy of hypertensive patients [4]. Bradykinin has vascular effects through stimulation of nitric oxide production [5]. Accumulation of endogenous bradykinin is involved in the vascular effects of ACE inhibitors in humans [6]. However, the exact contribution of bradykinin to the beneficial effects of ACE inhibition on cardiovascular hypertrophy is still uncertain [7].

Regulation of ACE is only partly understood. A number of compounds have been reported to increase ACE expression or activity in cultured endothelial cells. Those compounds include endothelin-1 [8], dexamethasone and certain other steroids [9], and platelet activating factor [10]. Induction of ACE by captopril in cultured human endothelial cells has been reported [11]. Cyclic nucleotide analogs, dibutyryl cyclic AMP and dibutyryl cyclic GMP stimulate ACE activity in bovine pulmonary artery endothelial cells [12]. Inhibition of ACE activity by lipopolysaccharides in human umbilical cord vein endothelial cells (HUVEC) has been reported [13]. Previous findings suggest that several independent intracellular signalling mechanisms regulate ACE, including cyclic AMP, Ca2+, G proteins, and Na+K+-ATPase.

In the present study regulation of ACE by atrial natriuretic peptide (ANP) and the role of cyclic GMP in ACE regulation was studied in cultured human endothelial cells.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Endothelial cell culture
With the approval of the committee of Ethics of the Department of Obstetrics and Gynecology, University of Helsinki, endothelial cells were prepared from human umbilical cord veins according to Jaffe et al. [14]. Veins were cannulated, washed with phosphate buffered saline (PBS) and treated with 0.5% collagenase (Sigma,St.Louis,MO,USA) in PBS for 15 min at room temperature and then collected by centrifugation. Cells were grown to confluence in 0.2% gelatin (Sigma) coated cell culture flasks (Costar, Cambridge,USA) in Medium 199 (Gibco Laboratories Inc., Belmont, California, USA) supplemented with 20% fetal calf serum (Gibco), 20 µg/ml endothelial cell growth supplement (Sigma), 12 U/ml heparin (Sigma), 100 U/ml G-penicillin, 100 µg/ml streptomycin (Gibco) and 2 mM L-glutamine (Gibco) at 37°C in humidified 5% carbon dioxide in air. The cells were detached with 0.125% trypsin–0.02% Na2EDTA solution (Gibco) and subcultured on 48-well cell culture plates (Costar) coated with 0.2% gelatin solution.

2.2 Experimental design
Confluent subcultures (at passages 1–2) were incubated for 24 hours with Medium 199 supplemented with 5% fetal calf serum with or without the following substances added in one dose: Atrial natriuretic peptide (ANP, 0.3–30 nM), Rp-8-Br-PET-cGMPS (1 µM), 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP, 1–100 µM), zaprinast (1–100 µM), dipyridamole (1–10 µM), 3-isobutyl-1-methylxanthine (IBMX, 0.1–0.5 mM), or cycloheximide (0.2–2 µg/ml). Cell cultures were preincubated with Rp-8-Br-PET-cGMPS for 15 min before adding ANP and with cycloheximide for 30 min before adding test substances. After incubation time ACE assay was performed as described below. The effect of test substances on cellular viability and growth were tested by a CellTiter 96 cell proliferation/cytotoxicity assay kit (Promega, Madison, Wis,USA). Rp-8-Br-PET-cGMPS was from Biolog Life Science Institute (Bremen, Germany), other substances were from Sigma.

2.3 cGMP measurement
At confluence endothelial cells were incubated for 30 min with ANP (3–30 nM), zaprinast (10–100 µM), IBMX (0.1–0.5 mM), or dipyridamole (10–100 µM), in Medium 199 supplemented with 5% fetal calf serum. After incubation the medium was aspirated and the cells scraped from culture dishes and suspended in 0.1 M HCl. The cell suspensions were snap-frozen and stored at –70°C until measurement of cGMP. The cell suspensions were centrifuged, and cGMP measured from neutralized supernatants using cGMP RIA kits (Amersham, Buckinghamshire, UK).

2.4 ACE inhibitor binding assay
ACE amount in intact endothelial cells was measured by a modified method of an inhibitor binding assay (IBA) developed in our laboratory [15], and calibrated with the method of Watanabe et al [13], using hippuryl-histidyl-leucine (Sigma) as a substrate. A lisinopril analog, 351A=(p-hydroxybenzamidine derivative of N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) a gift of Dr. M. Hichens (Merck, Sharp and Dohme, Rahway, NJ, USA), was labeled with 125-iodine (IMS 30, Amersham, Bucks, England) using chloramine T method, as described elsewhere [15]. 125I-351A is specifically bound to ACE[15, 16]. Thus, bound label is displaced from ACE binding by unlabeled lisinopril (0.125–2 ng/ml, Merck, Sharp and Dohme), and ramiprilate (0.125–2.5 ng/ml, Hoechst, Frankfurt am Main, Germany). After incubation of HUVECs with test substances cell monolayers were washed with PBS. Typically, 10 000 cpm of label per well in culture medium was added to HUVEC's cultured on 48-well plates. Following incubation at 37°C for 2 h, cells were washed twice with PBS, then detached with 0.1 M NaOH, and counted in a gamma counter.

2.5 Statistical evaluation
Results are expressed as mean ± SEM of six replicate determinations from three to four separate experiments. Student's t-test for paired or unpaired observations was applied.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Assay characteristics
Membrane bound ACE could be determined in intact endothelial cells grown on 48-well plates. The amount of ACE used as a measure of ACE throughout this report is expressed as a measure of the quantity of bound ACE inhibitor, 125I-351A (cpm/105 cells), previously shown to correspond to enzyme activity [13]. The dose dependent displacement of labeled ACE inhibitor from endothelial cells with lisinopril and ramiprilate (Fig. 1) and the Scatchard analysis (Fig. 2) show the specificity of the IBA assay. Non-specific binding was <1%.


Figure 1
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  		Fig. 1 Displacement curve of 125I- labelled lisinopril analog, 351A, with lisinopril and ramiprilate in intact human endothelial cell culture.

 

Figure 2
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  		Fig. 2 Scatchard analysis for 125I-351A binding to HUVEC. Increasing concentrations of 125I-351A were incubated for 2 h with intact endothelial cell culture at 37°C in humidified 5% carbon dioxide in air in the presence or absence of 100 nM lisinopril.

 
3.2 Stimulation of ACE
Incubation of endothelial cells with human ANP (0.3–30 nM) dose dependently increased ACE (Fig. 3a). The stimulatory effect of ANP was blocked by protein kinase G inhibitor, Rp-8-Br-PET-cGMPS (Fig. 3b). cGMP analog, 8-Br-cGMP (1–100 µM), dose dependently increased ACE (Fig. 4). The selective cGMP phosphodiesterase, type V inhibitor, zaprinast (10–100 µM), and the nonselective phosphodiesterase inhibitors, dipyridamole (1–10 µM) and IBMX (0.1–0.5 mM) all increased ACE (Fig. 5). The stimulatory effects of all the test substances were blocked by cycloheximide (0.2 and 2 µg/ml) (data not shown) indicating that de novo protein synthesis was needed.


Figure 3
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  		Fig. 3 Increase of ACE in endothelial cells treated with ANP (a). Effect of cGMP antagonist=Rp-8-Br-PET-cGMP (1 µM) on basal and ANP stimulated ACE amount (b). Rp-8-Br-PET-cGMP was added 15 min before ANP. Incubation time was 24 h. Bars indicate S.E.M. *p<0.05, ** p<0.01, *** p<0.001, compared to control.

 

Figure 4
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  		Fig. 4 Dose dependent increase of ACE in endothelial cells treated with 8-Br-cGMP after 24 h incubation. Bars indicate S.E.M. ** p<0.01, *** p<0.001 compared to control.

 

Figure 5
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  		Fig. 5 Effect of zaprinast (a), dipyridamole (b), and IBMX (c), on ACE after 24 h incubation. Bars indicate S.E.M. ** p<0.01, *** p<0.001, compared to control.

 
3.3 Stimulation of cGMP
Enhanced intracellular cGMP levels were measured after 30 min incubation time with ANP (3–30nM), and the phosphodiesterase inhibitors zaprinast (10–100 µM), IBMX (0.1–0.5mM), and dipyridamole (10–100 µM) (Table 1).


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  		Table 1 Effects of ANP, zaprinast, IBMX and dipyridamole on cGMP levels of cultured human umbilical vein cells

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
ANP, a diuretic, natriuretic, and vasodilatory peptide was shown here to increase the amount of ACE in intact HUVEC monolayers. The concentrations of ANP capable of inducing ACE were within the range observed in congestive heart failure [17], and may thus be considered biologically relevant.

ANP acts via two main types of specific receptors; guanylate cyclase linked receptors and a receptor thought to have a clearance function. Binding of ANP to guanylate cyclase linked receptors leads to increased intracellular cGMP levels [18]. The intracellular action of ANP involves increased cGMP levels.

To study the role of cGMP as an intracellular regulator of ACE an analog of cGMP, 8-Br-cGMP was used. 8-Br-cGMP increased ACE in intact HUVEC monolayers. Increase of ACE was also observed using a selective cGMP phosphodiesterase inhibitor, zaprinast, which increases intracellular cGMP levels by inhibiting breakdown of cGMP. Further, the cGMP antagonist, RP-8-Br-PET-cGMPS, inhibited ACE induction by ANP. These data suggest that cGMP is an intracellular regulator of ACE and that induction of ACE by ANP involves elevated intracellular cGMP levels. No effect on basal ACE amount was observed with the cGMP antagonist. This suggests that ACE is insensitive to modulation by low levels of cGMP spontaneously produced by endothelial cells.

In contrast to these observations, inhibition of ACE activity by ANP in cultured bovine aortic endothelial cell line has been reported [19]. This controversy may be explained by different sources of cells used. Conflicting results concerning ACE regulation in endothelial cell cultures have been reported, which suggests that there may be differences in ACE regulation depending on the origin of endothelial cells. For example phorbol ester (PMA) did not modify ACE activity in bovine pulmonary artery endothelial cells [20]whereas in HUVEC PMA is a potent stimulator of ACE [21](and unpublished results from our laboratory).

To study further the role of cyclic nucleotides in ACE regulation, two nonselective phosphodiesterase inhibitors, dipyridamole and IBMX, were used. Both of these compounds increased ACE amount. These phosphodiesterase inhibitors inhibit various isoenzymes of the phosphodiesterase family, and thus also inhibit degradation of cAMP [22]. As dibutyryl cyclic AMP and activators of adenylate cyclase, forskolin and cholera toxin, also stimulate ACE in HUVEC (unpublished observations from our laboratory), increased ACE by the nonselective phosphodiesterase inhibitors may partly be a result of increased intracellular cAMP. Therefore, compounds which regulate cyclic nucleotide synthesis or degradation by regulating phosphodiesterases are possible regulators of ACE.

In summary, the present data suggest that cGMP is a mediator involved in ACE regulation. ANP increased ACE possibly by increasing intracellular cGMP. ANP and cGMP mediate vasodilation, while ACE, by causing increased production of angiotensin II, promotes vasoconstriction. It may be concluded that substances increasing ANP and cGMP would lead to increased ACE expression as a counterbalancing mechanism on the cellular level. The interaction of ANP and ACE may represent an additional mechanism for vascular control.

Time for primary review 21 days.


   Acknowledgements
 
Supported by grants from the Sigrid Jusélius Foundation (Helsinki), the Finnish-Norwegian Medical Foundation (Helsinki), the Aarne Koskelo Foundation, the Finnish Heart Association, the Liv och Hälsa Foundation and Helsinki University Central Hospital Research Funds.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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