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Cardiovascular Research 2004 61(4):724-735; doi:10.1016/j.cardiores.2003.12.003
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Arginine–aminopeptidase in rat cardiac fibroblastic cells participates in angiotensin peptide turnover

V.V Petrov, R.H Fagard and P.J Lijnen*

Hypertension and Cardiovascular Rehabilitation Unit, Campus Gasthuisberg, Department of Molecular and Cardiovascular Research, University of Leuven (KULeuven), Herestraat 49, Louvain 3000, Belgium

* Corresponding author. Tel.: +32-16-34-57-66; fax: +32-16-34-62-03. victor.petrov{at}med.kuleuven.ac.be

Received 15 May 2003; revised 2 December 2003; accepted 3 December 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The aim of the present study was to elucidate the presence in rat cardiac fibroblastic cells of arginine–aminopeptidase and its involvement in the hydrolysis of angiotensin peptides. Methods: Peptidase activity was measured as hydrolysis of the synthetic substrates, aryl-p-nitroanilides. Immunoblottings were performed with antibodies to aminopeptidase B and Glyceraldehyde-3-phosphate dehydrogenase. Results: Arginine–aminopeptidase found in cardiac fibroblasts (Fb) was arginine and lysine specific, sensitive to various aminopeptidase (AP) inhibitors and to the inhibitor of metalloproteases, 1.10-phenatroline. Experiments with arphamenine A, a specific inhibitor of aminopeptidase B, have shown the presence of two Arginine–aminopeptidase activities: arphamenine-sensitive: chloride-stimulated Arginine–aminopeptidase, and arphamenine-insensitive: chloride-insensitive Arginine–aminopeptidase. Transforming growth factor-β1 stimulated both Arginine–aminopeptidase activities by approximately threefold. Immunoblot with an antibody specific to rat aminopeptidase B has revealed that arphamenine-sensitive: chloride stimulated aminopeptidase is aminopeptidase B. Arginine-p-nitroanilide hydrolysis was significantly inhibited by angiotensin peptides such as angiotensin (1–10), (1–8), (1–7), (1–4), (5–8), (4–8), (3–8), and (2–8) at the concentration of 50 µmol/l which was fourfold less than the Arginine-p-nitroanilide concentration. Conclusions: Our data show that chloride-insensitive Arginine–aminopeptidase could contribute to the hydrolysis of all studied angiotensin peptides in concert with other peptidases present in fibroblasts. Some of the peptides could probably not be hydrolyzed by Arginine–aminopeptidase. Instead, they could be first hydrolyzed by another peptidase in fibroblasts and the product of this hydrolysis could be a substrate for Arginine–aminopeptidase. The data obtained suggest that Arginine–aminopeptidase could perform processing of angiotensin peptides in the myocardium and participate in processes regulated by angiotensins such as fibrosis.

KEYWORDS Fibroblasts; Aminopeptidase B; Angiotensin peptides; Transformic growth factor-β


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Rat cardiac fibroblastic cells contain angiotensin converting enzyme (ACE) and secrete angiotensin II (ANGII) into the extracellular space [1–3]. Cathepsin D, as well as ACE, performs conversion of ANGI to ANGII in rat fibroblasts (Fb) by cleaving the Phe8–His9 bond [3]. Other peptidases, such as endopeptidases and carboxypeptidases, degrade angiotensin peptides by cleaving bonds different from the Phe8–His9 bond [4,5]. Rat chymase, in contrast to human chymase, has a very low activity, if any, for the conversion of ANGI to ANGII [6–8]. However, rat chymase efficiently cleaves the Tyr4–Ile5 bond in ANGI forming biologically inactive peptides [9–11]. It has been concluded that rat heart ACE is the main, if not the only, contributor to the conversion of ANGI to ANGII [7].

Processing of angiotensin peptides can also be performed by aminopeptidases (AP). AP-A digest Asp1–Arg2 in ANGI and ANGII [5,12–14]. AP-N digest Asp1–Arg2 in ANGI and Arg2–Val3 in ANGIII [4,5,15,16]. AP contribute to the control of plasma and tissue level of ANGI, ANGII, ANGIII, and ANG(1–4) [17,18]. Little is known about Arg–AP. However, it was shown that a particular type of Arg–AP, AP-B, hydrolyzes ANGI, and that treatment with AP-B inhibitors suppressed the development of hypertension in spontaneously hypertensive rats [19].

Previously, we have found that TGF-β1 stimulates expression of ACE and its activity during differentiation of cardiac fibroblasts to myofibroblasts (MyoFb) [2]. ACE activity was studied by liberation of the dipeptide His–Leu from a synthetic substrate Hippuril–Histidil–Leucine (Hip–His–Leu). It was surprising that only 30% of the Hip–His–Leu hydrolysis was due to true ACE activity. We have not yet identified peptidase(s) which perform the hydrolysis of Hip–His–Leu in concert with ACE. In addition, we have found that the dipeptide His–Leu was hydrolyzed which caused an underestimating of Hip–His–Leu hydrolytic activity. The hydrolysis of His–Leu was prevented in the presence of a broad range of aminopeptidase inhibitor, bestatin. These results allowed us to suggest that certain AP could be involved in the turnover of the angiotensin peptides in rat cardiac Fb. The aim of the present study was to elucidate the possible presence of Arg–AP in rat cardiac ventricular Fb and its involvement in the angiotensin peptide processing. Effect of the TGF-β1-induced differentiation of Fb to MyoFb on the Arg–AP expression and activity was also investigated.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell cultures
All animal procedures were in accordance with the laws, regulations and administrative provisions of the Member States of the European Community (Council Directive 86/609/EEC of November 24, 1986) regarding the protection of animals for experimental and other scientific purposes. This research protocol was also approved by the Ethical Committee for Animal Experiments of the Katholieke Universiteit Leuven (KULeuven, Belgium).

Cardiac ventricular fibroblasts were obtained from male Wister rats, 7 to 8 weeks old and weighing about 200 g. Primary cultures of cells prepared from the cardiac ventricles were cultured for 3 days until confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 75 cm2 tissue culture flasks. Cells of the first passage were plated at a density of 2600 cells/cm2 and cultured for 3 days until confluence in DMEM in the presence of 10% FBS. In the present study, only fibroblasts from passage two were used. For the second passage, cells were also plated at a density of 2600 cells/cm2 and cultured in DMEM in the presence of 10% FBS. After 24 h, the medium was replaced with fresh DMEM with 10% FBS containing TGF-β1 400 pmol/l and incubated for 7 days as previously described [2].

The cultures were characterized by staining with antivimentin, antidesmin, antifactor VIII, and anti-{alpha}-smooth muscle actin ({alpha}-SMA) antibodies [2,20]. Control cultures consisted of approximately 86% of Fb and 14% of MyoFb while TGF-β1-treated cultures consisted of almost 100% of MyoFb [2].

2.2 Immunoblot
Western blotting for the determination of {alpha}-SMA has been performed as previously described [2,20]. Aminopeptidase B was examined with a specific rabbit anti-AP-B polyclonal antibody generously provided by Dr. P. Cohen (Laboratoire de Biochemie des Signaux Régulateurs Cellulaires et Moleculaires, Université Pierre et Marie Curie, Paris, France). The antibodies were raised in rabbits for the purified rat testes AP-B [21]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was examined with a specific mouse monoclonal antibody (Biogenesis, UK).

2.3 Enzyme activity
AP activity was determined with synthetic substrates L-aminoacyl–para-nitroanilides (aminoacyl–pNA), such as Arg–, Ala–, Lys–, and Gly–pNA at a concentration of 1 mmol/l according to the method of Balogh et al. [22]. In experiments with angiotensin peptides, the concentration of Arg–pNA was 200 µmol/l. The reaction was linear with respect to incubation time and to enzyme concentration for at least 2 h. The concentration of the angiotensin peptides, if not additionally mentioned, was 50 µmol/l.

Total AP activity (T) was measured in homogenates of Fb. After incubation of cell homogenates with phosphate buffered saline (PBS, containing 9.3 mM Na2PO4, 2.9 mM K2PO4, 3 mM KCl, and 136 mM NaCl, pH 7.2) in the presence of substrate for 1 h at 37 °C, the concentration of released nitroaniline was determined at an absorbance at 405 nm. The reaction was stopped by the addition of sodium acetate (1 M, pH 4.2). To measure AP activity on intact Fb, cells were grown to confluence, washed with phosphate buffer, and then incubated for 1 h with buffer containing substrate at a concentration of 1 mmol/l. Subsequently, a concentration of free p-nitroaniline was determined in the buffer after its aspiration and centrifugation.

In these experiments, two activities of Arg–pNA hydrolysis (M+S) were measured together: activity performed by the membrane-bound enzyme (M) and activity performed by the enzyme secreted into the medium (S). To discriminate between membrane-bound (M) and secreted Arg–AP (S), cell cultures were also incubated with PBS in the absence of the substrate. After incubation for 1 h, buffer was removed from the culture and centrifuged to eliminate the suspended cells. Then, the substrate Arg–pNA was added to the buffer, and the enzyme activity was measured after incubation for 1 h. This activity was performed by enzyme secreted into the medium (S). All measured activity was calculated per 106 cells. Membrane Arg–AP was estimated as the difference [(M+S)–S] between whole enzyme activity in medium (M+S) and secreted enzyme activity (S). Intracellular activity was calculated as the difference (T–M) between total activity in homogenates (T) and membrane-bound activity (M) in Fb.

Other substrates of AP, aminoacyl–β-naphtylamides (aminoacyl–βNA), such as Ala–, Arg–, and Lys–βNA were used as competitive inhibitors of aminoacyl–pNA hydrolysis.

2.4 Statistical analysis
Values were expressed as mean±S.E.M. The statistical methods used were repeated measures of variance (Tukey's), or Student's two-tailed test for paired data when appropriate. P-value<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Hydrolysis of various L-{alpha}-aminoacyl–p-nitroanilides
Homogenates of control cultures of cardiac Fb hydrolyzed synthetic aminopeptidase substrates such as Arg–pNA, Ala–pNA, Leu–pNA, Lys–pNA, and Gly–pNA (Table 1). Degradation of Arg–pNA, Ala–pNA, Leu–pNA, and Lys–pNA was totally inhibited in the presence of 1 mmol/l of 1,10-phenantroline (data not shown), a ligand for polyvalent metal cations. Bestatin (100 µmol/l), an inhibitor with broad specificity for aminopeptidases, inhibited the hydrolysis of Ala–pNA and Arg–pNA by approximately 90%, and Lys–pNA and Leu–pNA by 70% and 40% respectively, indicating that peptidases degrading these substrates were aminopeptidases (AP) which could be attributed to the metalloproteases. However, neither 1,10-phenantroline nor bestatin inhibited the hydrolysis of Gly–pNA, demonstrating that this substrate is a target for a peptidase which is not a metallopeptidase or an AP.


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  		Table 1 Rate of hydrolysis of various L-{alpha}-aminoacyl–p-nitroanilides by homogenates of control or TGF-β1-treated fibroblasts in the absence or presence of bestatin

 
3.2 Stimulation of APs activity by TGF-β1
Previously, we have demonstrated that TGF-β1 induced a differentiation of Fb to MyoFb resulting in an increase of the amount of MyoFb in the cultures from 14% to 100% [2,20]. Table 1 shows that TGF-β1 in the same conditions stimulated (by approximately two- to threefold) the hydrolytic activity of Ala–pNA, Leu–pNA, Lys–pNA, and Arg–pNA performed by AP, as well as Gly–pNA hydrolysis performed by peptidase different from AP.

3.3 Inhibitors of the Arg–pNA hydrolysis
To verify which AP degrade Arg–pNA, the effects of various AP inhibitors that are specific for different peptidases were studied.

Peptidase inhibitors with a broad specificity such as aprotinin (10 µmol/l), leupeptin (10 µmol/l), or pepstatin A (100 µmol/l); E-64 (100 µmol/l; inhibitor of cathepsin H); chymostatin (100 µmol/l; inhibitor of chymase); imipramine (300 µmol/l; inhibitor of enkephalinase); phosphoramidon (10 µmol/l; inhibitor of endopeptidase 24.11); lisinopril (10 µmol/l; inhibitor of ACE); and apstatin (10 µmol/l; inhibitor of AP-P) did not change Arg–pNA hydrolysis significantly (data not shown).

Bestatin, a broad range AP inhibitor [23–25], inhibited Arg–AP up to 10% of the basal level at a concentration of 300 µmol/l. TGF-β1 decreased this inhibition (Fig. 1A).


Figure 1
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  		Fig. 1 Effect of the aminopeptidase inhibitors bestatin (A), actinonin (B), amastatin (C), arphamenine A (D), leuhistin (E), and puromycin (F) on the activity of Arg–pNA hydrolysis in control or TGF-β1-treated Fb. Reactions were carried out in phosphate buffer containing 150 mmol/l NaCl (pH 7.2). Concentration of Arg–pNA was 1 mmol/l.

 
Puromycin, a potent inhibitor of Ala–AP and a specific inhibitor of a particular Ala–AP type, AP-S, or puromycin-specific AP [26–29], did not inhibit Arg–AP activity significantly up to a concentration of 1 mmol/l. Puromycin slightly inhibited Arg–pNA hydrolysis in TGF-β1-treated cells (Fig. 1F).

Amastatin, a specific inhibitor of various types of the Ala-specific APs, such as AP-N (known also as AP-M or CD13) [30] and AP-S [29], and a potent inhibitor of Leu–AP [31,32] inhibited Arg–pNA hydrolysis up to approximately 50% of the basal level in the control cells and up to 70% in the TGF-β1-treated Fb at a concentration of 300 µmol/l (Fig. 1C).

Actinonin and leuhistin, specific inhibitors of Ala–AP [30,33,34] inhibited Arg–pNA hydrolysis up to 40% at a concentration of 300 µmol/l, independently of the treatment with TGFβ1 (Fig. 1B and E).

Arphamenine A, a specific inhibitor of Arg–AP, particularly AP-B [21,35–37] inhibited Arg–pNA hydrolysis dose-dependently. A maximal inhibition of 40% was found at a concentration of 1 µmol/l. Further increase of the inhibitor concentration up to 1 mmol/l did not induce an additional inhibition of Arg–AP. These results indicate the presence in Fb of two activities of hydrolysis: arphamenine-sensitive and arphamenine insensitive. Arphamenine inhibited the hydrolysis of Arg–pNA to a lesser extent in the TGF-β1-treated cells (Fig. 1D).

Arg–pNA hydrolysis could be performed by Arg–AP which highly specifically degraded basic arylamides (Arg–pNA and Lys–pNA) [21,35,38–41], or by Ala–AP and AP-S. The latter two AP are not specific and hydrolyze Arg–amide as well as Ala–amide although with a lower activity than Ala–pNA [15,16,26,29,35,42]. To elucidate if Arg–pNA could be hydrolyzed by Ala–AP, AP-S, or Leu–AP, the inhibitory effects of amastatin, leuhistin, and puromycin were compared with two substrates, Ala–pNA and Arg–pNA. Amastatin and leuhistin almost completely inhibited Ala–pNA hydrolysis at a concentration of 3 µmol/l (Fig. 2), while Arg–pNA hydrolysis was inhibited by 50% at a concentration of 300 µmol/l (Fig. 1C and E). The most obvious difference was found with puromycin which, at a concentration of 1 mol/l, almost completely inhibited Ala–pNA hydrolysis (Fig. 2), while there was no significant inhibition of Arg–pNA (Fig. 1F).


Figure 2
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  		Fig. 2 Dose-dependent inhibition of Ala–pNA hydrolysis by amastatin, leuhistin, and puromycin. Reactions were carried out in phosphate buffer containing 150 mmol/l NaCl (pH 7.2). Concentration of Ala–pNA was 1 mmol/l.

 
3.4 Competition between aminoacyl–pNA and aminoacyl–βNA
The competition between the substrates, Arg–amide, Ala–amide, and Lys–amide was also studied to examine the possible contribution of Ala–AP and/or Leu–AP in the Arg–pNA hydrolysis. Ala–βNA did not inhibit Arg–pNA hydrolysis significantly in a concentration range of 0 to 1 mmol/l, while Arg–βNA dose-dependently inhibited the hydrolysis of Ala–pNA (Fig. 3A). TGF-β1 did not change the competition between these substrates (Fig. 3B). Lys–βNA, in contrast to Ala–βNA, dose-dependently inhibited Arg–pNA hydrolysis (Fig. 3C).


Figure 3
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  		Fig. 3 Inhibition of aminoacyl–p-nitroanilide hydrolysis in homogenates of control Fb (A) or TGF-β1-treated Fb (B) by aminoacyl–βNA. (C) Inhibition of Arg–pNA hydrolysis by Lys–βNA in control Fb. All reactions were carried out in phosphate buffer containing 150 mmol/l NaCl (pH 7.2). Concentration of aminoacyl–pNA was 1 mmol/l.

 
3.4.1 Effect of chloride
3.4.1.1 Total Arg–AP activity
In homogenates of Fb, NaCl dose-dependently (50–150 mmol/l) stimulated Arg–pNA hydrolysis (data not shown). At a concentration of 150 mmol/l, NaCl significantly stimulated the hydrolysis by approximately 50% as compared to phosphate buffer containing Na+ 145 mmol/l in the absence of Cl– (Fig. 4A). These data suggest that AP, which performed the hydrolysis of Arg–pNA, resembles Cl–-sensitive AP-B (EC 3.4.11.6) that specifically liberates basic amino acids arginine and lysine from the N-terminus of synthetic substrates, L-{alpha}-aminoacylamides. The Lys–pNA hydrolysis was also significantly stimulated by Cl–, although the effect was less pronounced (14.5±1.0%, p<0.01) as compared to Arg–pNA.


Figure 4
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  		Fig. 4 (A–B) Effects of NaCl and arphamenine A on Arg–pNA hydrolysis in homogenates of control fibroblastic cells (A) and TGF-β1-induced MyoFb (B). Reactions were carried out either in phosphate buffer alone or in phosphate buffer containing NaCl 150 mmol/l. Concentrations of Arg–pNA were 1 mmol/l, and of arphamenine 100 µmol/l. **: p<0.01; ***: p<0.001 as compared to basal; ++: p<0.01; +++: p<0.001 as compared to basal without NaCl; ooo: p<0.001 as compared to basal in control Fb. (C) Immunoblot of AP-B and GAPDH in homogenates of control and TGF-β1-treated Fb. (D) Calculated arphamenine-sensitive Arg–pNA hydrolysis in the presence of NaCl in control Fb (A) and in TGF-β1-treated Fb (B). ***: p<0.001 as compared to Control.

 
3.5 Arphamenine-sensitive Arg–AP
Arphamenine A, a specific inhibitor of AP-B, at a concentration of 100 µmol/l, slightly but significantly reduced the activity of Arg–pNA hydrolysis in homogenates of control Fb in the absence of Cl– [from 0.89±0.11 to 0.78±0.11 nmol/(min x 106 cells), p<0.01]. The inhibitory effect of arphamenine was much more pronounced in the presence of Cl– (from 1.45±0.10 to 0.74±0.08, p<0.001; Fig. 4A). Thus, arphamenine-sensitive Arg–pNA hydrolysis was stimulated 6.5-fold by Cl–. However, the level of the hydrolysis activity in the presence of arphamenine (arphamenine-insensitive) was not significantly changed by Cl– (Fig. 4A), suggesting that arphamenine-insensitive Arg–AP is Cl–-insensitive as well.

In TGF-β1-treated cultures as well as in control cultures of Fb, the level of arphamenine-sensitive Arg–pNA hydrolysis was stimulated by fivefold (from 0.40±0.06 to 2.05±0.07) by Cl–, while no stimulation of the arphamenine-insensitive Arg–AP activity was found (Fig. 4B).

Expression of {alpha}-smooth muscle actin in TGF-β1-treated cultures of Fb, which consisted of almost 100% of MyoFb, was 2.6-fold higher than in control cultures of Fb (data not shown). This stimulation was accompanied by Arg–AP activity stimulation (approximately threefold) independently of the presence of Cl– (Fig. 4B). Arphamenine-sensitive Arg–AP in the presence of Cl– was increased from 0.63±0.03 to 2.02±0.06 (Fig. 4D).

3.6 Immunoblot of AP-B
The specific hydrolysis of Arg–pNA, the inhibitory effect of arphamenine, the stimulation of the hydrolysis of Arg–pNA by Cl–, and the dramatic stimulation of the arphamenine-sensitive activity by Cl– suggest that AP-B could contribute to the Arg–pNA hydrolysis in homogenates of Fb and MyoFb.

Immunoblotting with antibodies specific for AP-B revealed the presence of AP-B in homogenates of control Fb. Expression of AP-B in TGF-β1-treated cultures of Fb was increased (p<0.5) by 3.6±0.8-fold, while expression of GAPDH used as a sample control loading was not changed (percent change in TGF-β1-treated Fb from control cultures was 0.4±2.7%, p = 0.45; Fig. 4C).

3.7 Localization of Arg–AP
The total Arg–AP activity in Fb consisted of the intracellular (36%) and membrane-bound activity (46%; Fig. 5). The difference between cytoplasmic and membrane enzyme activities was not significant. Little activity was found in the conditioned medium after incubation for 1 h. Dead cells as assayed by trypan blue exclusion were not found in the cultures just before and after 1 h incubation of the cells with phosphate buffer. Therefore, we believe that this activity belongs to the Arg–AP that was secreted from Fb. Secreted Arg–AP activity comprised 12% of total activity or 30% of the cytoplasmic activity.


Figure 5
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  		Fig. 5 Distribution of Arg–pNA activity hydrolysis in Fb. Intracellular, membrane-bound, and secreted forms of Arg–AP. Arg–pNA hydrolysis was performed in homogenates and in cultures of intact Fb. Measurement and calculation of the Arg–AP are described in Methods. Reactions were carried out in phosphate buffer containing 150 mmol/l NaCl (pH 7.2). Concentration of Arg–pNA was 1 mmol/l.

 
3.8 Inhibition of Arg–AP with angiotensin peptides
ANGII dose-dependently inhibited the hydrolysis of Arg–pNA (Fig. 6B). At an ANGII concentration of 200 µmol/l, inhibition of Arg–AP activity averaged 58.5±3.7% (p<0.001). This inhibition could be explained by the competition between Arg–pNA and ANGII.


Figure 6
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  		Fig. 6 Effect of angiotensin peptides on Arg–pNA hydrolysis in homogenates of Fb. Concentration of Arg–pNA was 200 µmol/l. (A) Effect of ANGII. ANGII was added either together with Arg–pNA and incubated for 0.5 h or preincubated for 1 h before addition of the Arg–pNA. Concentration of ANGII was 50 µmol/l. (B) Dose-dependent effect of ANGII. (C) Various angiotensin peptides. Concentration of peptides was 50 µmol/l.

 
Fig. 6A suggests that Arg–pNA-degrading AP could also hydrolyze ANGII. In control experiments, cell homogenates were preincubated for 1 h without Arg–pNA. After preincubation, Arg–pNA was added, and the homogenates were then further incubated for 0.5 h, and the liberation of nitroaniline from the substrate was measured. ANGII added simultaneously with Arg–pNA inhibited Arg–pNA hydrolysis by 30%. However, the inhibition disappeared if ANGII was added 1 h before Arg–pNA. This could be explained by a decrease in the concentration of ANGII during preincubation, which after 1 h was very low to inhibit Arg–pNA hydrolysis. The decrease in ANGII suggested a hydrolysis of the peptide in homogenates of Fb.

3.9 ANGII hydrolysis in TGF-β1-induced MyoFb
As shown in Fig. 7A, ANGII inhibited Arg–pNA hydrolysis either in the presence or in the absence of Cl–. Substrate hydrolysis was stimulated by TGF-β1. Fig. 7B shows ANGII-sensitive and ANGII-insensitive component of Arg–pNA hydrolysis by control Fb. There was no significant difference between ANGII-sensitive hydrolysis in the presence or absence of Cl–.


Figure 7
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  		Fig. 7 (A) Effect of Cl– on the ANGII-induced inhibition of Arg–pNA hydrolysis in Fb or TGF-β1-induced MyoFb. Reactions were carried out in the presence of either phosphate buffer alone or phosphate buffer containing NaCl 150 mmol/l. Concentrations of Arg–pNA were 200 µmol/l, and concentrations of ANGII were 50 µmol/l. Effect of Cl– on the ANGII-sensitive and ANGII-insensitive Arg–pNA hydrolysis. (B) Control Fb; (C) TGF-β1-induced MyoFb.

 
TGF-β1 did not stimulate significantly the ANGII-sensitive hydrolysis independently of the presence of Cl– (Fig. 7B and C). However, ANGII-insensitive Arg–AP activity was stimulated by TGF-β1 in the absence and in the presence of Cl– by six- and fivefold, respectively.

3.10 Angiotensin peptides
All studied angiotensin peptides inhibited Arg–pNA hydrolysis significantly (Fig. 6C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1 Substrate specificity of Arg–pNA hydrolyzing AP
Arg– and Lys–pNA hydrolysis observed in homogenates of Fb could be performed by Arg–AP (3.4.11.6, Cl–-activated Arg–AP, AP-B). This AP specifically removes basic amino acids, arginine, and lysine from the N-terminus of substrates [21,35,38–41,43,44]. Some other AP could also hydrolyze the Arg– and Lys–amide nonspecifically. To elucidate the type of AP, which degrades Arg– and Lys–amide in cardiac Fb, we examined AP substrate specificity.

Ala–AP, such as AP-N (EC 3.4.11.2) and/or AP-S (EC 3.4.11.1 [EC] 4, puromycin-sensitive AP), hydrolyze Ala–amide and Arg–amide with an activity ratio Ala:Arg in the range from 10:3 to 10:5 [15,16,26,29,35,42]. Ala–pNA hydrolysis, found in the current study (Table 1), may suggest the presence of Ala–AP in Fb. However, Ala–AP probably did not contribute to the Arg–pNA hydrolysis because puromycin, a potent inhibitor of Ala–AP and a specific inhibitor of AP-S [29], did not inhibit the Arg–pNA hydrolysis (Fig. 1F) while completely inhibiting the hydrolysis of Ala–pNA (Fig. 2).

4.2 Competition between Ala–βNA with Arg–pNA
To prove the lack of Ala–AP contribution to the Arg–pNA hydrolysis, competition between substrates containing either arginine or alanine was studied. We examined the inhibition of aminoacyl–pNA hydrolysis by aminoacyl–βNA (Fig. 3). Hydrolysis of aminoacyl–pNA and aminoacyl–βNA by AP depends on the kind of amino acid but not of amides, and liberated β-naphtylamine does not obstruct optical determination of liberated p-nitroaniline. Fig. 3A shows that Ala–βNA insignificantly inhibited (~12%) hydrolysis of Arg–pNA in homogenates of Fb. However, Arg–βNA inhibited the hydrolysis of Ala–pNA considerably (approximately threefold). These results can be explained by the presence in Fb of two AP: Arg–AP degrading specifically Arg–amide (with high rate), and less specific Ala–AP degrading both Ala–amide and Arg–amide with a high and a low rate, respectively.

It is possible to estimate an outcome of Ala–βNA on Arg–pNA hydrolysis. In the present study, Ala–pNA was hydrolyzed by Ala–AP with an activity of 0.58±0.08 (nmol nitroaniline)/(min x 106 cells) (Table 1). It has been previously shown that purified Ala–AP hydrolyzes Ala–amide with an activity two- to threefold higher than Arg–AP [15,16,26,29,35,42]. Thus, the activity of the Arg–pNA hydrolysis by Ala–AP in homogenates of Fb can be in the range of 0.19 to 0.29 (nmol nitroaniline)/(min x 106 cells) or approximately 17% of the total Arg–pNA hydrolysis activity (see Table 1). This is compatible with 12% inhibition of Arg–pNA hydrolysis by Ala–βNA as shown in Fig. 3A. The rest of the Arg–pNA hydrolysis activity (88%) belongs to Arg–AP. On the other hand, the substrate containing arginine strongly inhibited the hydrolysis of the substrate containing alanine. The threefold inhibition of Ala–pNA hydrolysis in the presence of Arg–βNA (Fig. 3A) can be explained by competition between the two substrates for the nonspecific Ala–AP only because Arg–AP cannot hydrolyze Ala–pNA [21,35,38–41].

Thus, Ala–AP in Fb removes alanine from substrates with much higher activity than arginine. This is compatible with the abovementioned data obtained with purified Ala–AP. Lys–βNA, in contrast to Ala–βNA, inhibited Arg–pNA hydrolysis, confirming specificity of the Arg–AP for basic amino acids, arginine and lysine (Fig. 3C).

4.3 Cl–-sensitive, arphamenine-sensitive AP hydrolyzing Arg–pNA
Like AP-B, purified from other cells, Arg–AP found in Fb was activated by Cl– (Fig. 4A). However, activation of Arg–AP in homogenates of Fb was moderate (1.5-fold stimulation) in contrast to the strong stimulation (5- to 20-fold) of purified AP-B [21,39,41,43,45]. The present study shows that two Arg–AP are present in rat cardiac Fb, namely arphamenine-, Cl–-insensitive AP and arphamenine-, Cl–-sensitive AP (Fig. 4A). Arphamenine-, Cl–-sensitive AP is responsible for only 10% of the total Arg–AP hydrolysis activity in the absence of Cl–. However, in the presence of Cl–, as much as half of the total activity belongs to this enzyme (Fig. 4A). Moderate stimulation of the total Arg–pNA hydrolysis by Cl– (1.5-fold), as compared with purified AP-B, can therefore be explained by the presence of two Arg–AP in Fb; one of them is strongly activated by Cl– (6.5-fold), and the other one is not.

Cl–-stimulated Arg–AP, representing only a part of the total activity of the Arg–pNA hydrolysis, is very similar to AP-B. Immunoblotting with antibodies specific to rat AP-B showed the presence of AP-B in rat cardiac Fb (Fig. 4C). Band of AP-B is positioned a little lower than the 76 kDa marker band. It is compatible with data obtained by others [21,38,41,43]. Therefore, we conclude that rat cardiac Fb contains AP-B, and that Cl–-stimulated arphamenine-sensitive Arg–pNA hydrolysis is performed by this enzyme.

4.4 Cl–-independent arphamenine-independent Arg–AP
The type of AP, which performed arphamenine- and Cl–-insensitive Arg–pNA hydrolysis, needs to be further elucidated. At present, we can only exclude some possible candidate peptidases. Leu–pNA hydrolysis found in cell homogenates suggests that Leu–AP (EC 3.4.11.1) could be present in Fb. However, this enzyme is not able to release ultimate Arg or Lys from the N-termini of peptides [46]. AP-P (EC 3.4.11.9 [EC] ), AP-H, and cathepsin H (EC 3.4.22.1 [EC] 6) are able to release ultimate arginine from N-termini of peptides. However, absence of inhibition of the Arg–pNA hydrolysis by apstatin, aprotinin, or NaCI suggests the absence of AP-P in Fb [47–49]. Strong inhibition of the hydrolysis by bestatin and lack of inhibition in the presence of leupeptin indicated the lack of AP-H [50]. Lack of hydrolysis inhibition by E-64 suggests the absence of cathepsin H [51,52].

4.5 Localization of Arg–AP
Fb have intracellular, membrane-bound, and secreted Arg–AP activity (Fig. 5). Despite low activity found in the conditioned medium, the secretion of Arg–AP is quite an active process because the activity of the secreted enzyme comprised 30% of the cytoplasmic activity already after 1 h of cell incubation. These data are compatible with a localization of Arg–AP in the membranes and cytoplasm as reported for other cell types [19,22,53]. Arg–AP was also shown to be secreted by cells in the extracellular space [22,32,36].

4.6 Angiotensin peptides are targets for Arg–AP
It was previously shown that AP-B hydrolyzes ANGI [43,45], and that treatment with AP-B inhibitors suppressed the development of hypertension in spontaneously hypertensive rats [19]. Therefore, we studied if angiotensin peptides can be substrates of Arg–pNA in cardiac Fb.

All angiotensin peptides, examined in the present study, were able to inhibit Arg–pNA hydrolysis. Data presented in Fig. 6A and B suggest that ANGII-induced inhibition of Arg–AP in homogenates of Fb can be explained by competition between the two substrates, ANGII and Arg–pNA. The affinity of Arg–AP to ANGII is high. Indeed, 50% inhibition of the Arg–pNA hydrolysis by ANGII was found at a concentration of 3 µmol/l while the concentration of Arg–pNA was 60-fold higher, i.e., 200 µmol/l. Cl– did not significantly increase ANGII-sensitive activity (Fig. 7B). Thus, at the present time, we can suggest that ANGII inhibited Cl–-independent Arg–AP, although Cl– sensitivity is not an absolute property of Arg–AP. Indeed, Arg–AP is Cl–-sensitive in the presence of the synthetic substrates, Arg– or Lys–amide. However, hydrolysis of peptide hormones by the Arg–AP was independent of Cl– [43,45].

Competition of the various angiotensin peptides with Arg–pNA (Fig. 7C) suggests that Arg–AP in Fb, besides arginine, could also release asparagine, valine, tyrosine, and isoleucine from the N-termini of various studied angiotensin peptides. However, we did not find hydrolysis of Asp–pNA, Val–pNA, and Ile–pNA in homogenates of Fb (data not shown). It does not mean, however, that Arg–AP can not remove these amino acids from the N-terminus of the peptides. Indeed, purified Arg–AP releases arginine and lysine highly specifically only from synthetic substrates [21,35,38,39,41]. On the other hand, the presence of Arg or Lys at the N-terminus was not an absolute requirement for dipeptide hydrolysis by the enzyme [41]. Moreover, it was found that the hydrolysis of some physiologically active peptides, including ANGI, occurred via cleavage of internal bonds, demonstrating endopeptidase activity of the Arg–AP [41,43,45]. To explain how Arg–AP could be involved in the hydrolysis of all studied angiotensin peptides, we should take into account that in homogenates of Fb various peptidases contribute to the angiotensin peptide hydrolysis such as aminopeptidases, endopeptidases, and carboxypeptidases [4,12–16]. Some particular peptide that is not a substrate of Arg–AP could be hydrolyzed by one of these peptidases, and a shorter peptide, appearing after hydrolysis, could be a substrate for Arg–AP competing with Arg–pNA. We suggest that Arg–AP is able to operate in concert with other peptidases and participates in the hydrolysis of all studied angiotensin peptides. Localization of Arg–AP in the plasma membrane permits us to suggest that this enzyme could play a role in the processing of angiotensin peptides in the myocardium. Stimulation of Arg–AP activity in TGF-β1-treated Fb suggests that this enzyme could play a role in cardiac fibrosis incidence. Experiments with a direct measurement of angiotensin peptide hydrolysis have to be further performed to elucidate which bonds of the peptides and which peptides can be hydrolyzed by Arg–AP.


   Acknowledgements
 
The authors gratefully acknowledge the technical assitance of Mrs. Tamara Coenen, Mrs. Lieve Lommelen, and Mrs. Yvette Piccart. This work was supported by an education grant of AstraZeneca (Belgium) and by a grant from the Fund for Scientific Research, Flanders (Belgium). Robert H. Fagard is a holder of the Professor Antoon Amery Chair in Hypertension Research founded by MSD (Belgium). Paul Lijnen is a holder of the Boehringer Ingelheim Chair in Hypertension.


   Notes
 
Time for primary review 28 days


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

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