Key words
blood pressure - renin-angiotensin-system - endothelium - tripeptide - cathepsin G
- prolyl oligopeptidase
Introduction
Blood pressure and vascular functions are regulated by numerous circulating and local
factors. The main regulatory system is renin-angiotensin system (RAS) ([Fig. 1]), but also other endothelial enzymes produce highly active compounds such as prostacyclin
and nitric oxide. The most biologically active substance in RAS is angiotensin II
(Ang II), which is broken down from angiotensin I (Ang I) mainly by angiotensin converting
enzyme I (ACE-1) but also by chymase and cathepsin G. ACE-1 also degrades bradykinin,
a vasodilatory peptide into inactive fragments ([Fig. 1]). RAS contains a number of other enzymes besides ACE-1 which have an important role
in blood pressure regulation. Some of these enzymes have a direct effect on Ang II
but they also participate in the conversion of substances such as angiotensin-(1–7)
(Ang-(1–7)) with potent vasodilatory and cardioprotective effects. [1]
[2]
[3] In addition to classical AT-1 receptors, new focus has also been set to vasodilatory
AT-type 2 and Ang-(1–7)-Mas-receptors [3]
[4]. Ang-(1–7) also increases the physiological impact of bradykinin. [3]
[4]
[5]
Fig. 1 Simplified cascade of the renin-angiotensin-system (RAS) and the bradykinin pathway.
Shaded boxes represent corresponding type of receptors.
When milk is fermented e. g. with L. helveticus or S. cerevisiae, casein is degraded into bioactive tripeptides isoleucine-proline-proline (IPP), leucine-proline-proline
(LPP) and valine-proline-proline (VPP). These tripeptides have been found to lower
blood pressure and improve vascular function in hypertensive rats [6]
[7]
[8]
[9] and in humans [10]
[11]
[12]. Their blood pressure-lowering effect is believed to result mainly from the inhibition
of ACE-1 activity [6]
[13], but some of their favorable vascular functions are different from those of captopril,
a standard ACE inhibitor [8]. This could be related e. g. to inhibition of arginase [13] which increases local substrate concentration for nitric oxide production.
The effects of the casein derived tripeptides cannot be explained solely by ACE-1
inhibition due to their manifold pharmacological effects. Therefore we investigated
their effects on other blood pressure and vascular function regulating enzymes in vitro.
Materials and methods
Tripeptides IPP, VPP and LPP, substrates dansyl-D-Ala-Gly-4-Nitro-Phe-Gly-OH (DAGNPG),
Z-Gly-Pro-AMC and N-Suc-Ala-Ala-Pro-Phe-pNA were purchased from Bachem (Weil am Rein,
Germany). NEP was purchased from Innovative Research (Novi, MI, USA). POP (Recombinant
Human), ECE-1 (Recombinant Human) and MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys-(DNP)OH
were purchased from R&D Systems (Minneapolis, MO, USA). Cathepsin G (Human neutrophil)
was purchased from Merck Chemicals (Nottingham, UK).
Renin assay
Renin (Human recombinant) activity was measured using a commercial fluorometric kit
(SensoLyte® 520 Renin Assay Kit, AnaSpec, San Jose, CA, USA). The activity of renin was determined
using 5-FAM/QXL™520 fluorescence resonance energy transfer (FRET) peptide as a substrate
which renin degrades into 2 separate fragments. The fluorescent can be monitored at
ex/em=490/520 nm (Perkin Elmer Life Science, WALLAC VICTOR2 1420, Turku; Finland). Incubation time was 60 min at 37°C including 11 fluorescent
measurements with all the tripeptides concentrations (1 µM-3.3 mM). The tripeptides
were solved and diluted in the assay buffer.
Chymase assay
Chymase (Human recombinant) activity was measured using commercial kit (Chymase activity
kit, Sigma-Aldrich, Jerusalem, Israel). The method is based on the ability of chymase
to degrade N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide into a colorimetric product which can be monitored at wavelength 405 nm
(Multiscan RC, Labsystems, Helsinki, Finland). Incubation time was 50 min at 37°C
including 9 measurements with all tripeptides concentrations (1 µM-1 mM). The tripeptides
were solved and diluted in the assay buffer.
Neutral endopeptidase (NEP) assay
NEP activity was measured using the method by Florentin et al. [14] with small modifications. Assay substrate was DAGNPG which was diluted first in
methanol and thereafter in assay buffer Tris-HCl (50 mM, pH 7.4), the final concentration
of the substrate in the reaction was 50 µM. Methanol concentration in the reaction
was 0.2%. The method was based on NEPs ability to degrade DAGNPG of which fluorescense
was monitored at ex/em=342/580 nm (Perkin Elmer Life Science, WALLAC VICTOR2 1420). Incubation time was 120 min at 37°C including 7 measurements with all tripeptides
concentrations (1 µM-1 mM). The tripeptides and the enzyme were solved and diluted
in the assay buffer.
Prolyl oligopeptidase (POP) assay
POP activity was measured using the method design by Stanziola et al. [15] and Atack et al. [16]. Assay substrate was Z-Gly-Pro-AMC from which POP can cleave AMC group, a fluorescent
substance which can be measured in ex/em=355/460 nm (Perkin Elmer Life Science, WALLAC
VICTOR2 1420). The assay buffer was Tris (25 mM)-NaCl (250 mM)- dithiotreitol (DTT, 2.5 mM)
buffer (pH 7.5). The enzyme, the tripeptides and the substrate were diluted in the
assay buffer. The final concentrations of the enzyme, the substrate and the tripeptides
in reaction were 0.005 µg, 50 µM and 1 µM-3.3 mM, respectively, and the incubation
time was 60 min, including 10 measurements at room temperature.
Cathepsin G assay
Cathepsin G activity was measured using the method by Barret [17] with a small modifications. The method is based on the ability of cathepsin G to
degrade the substrate N-Suc-Ala-Ala-Pro-Phe-pNA into a colorimetric product that can
be measured spectrophotometrically at 410 nm (Multiscan RC, Labsystems, Helsinki,
Finland). The substrate was diluted in dimethyl sulfoxide (DMSO). The final concentration
of the substrate and DMSO in the reaction was 200 µM and 25%, respectively. The enzyme
(0.1–0.2 mU/reaction) was diluted in ice cold distilled water. The tripeptides were
solved and diluted in HEPES-NaOH buffer (100 mM, pH 7.5) which was the assay buffer.
The incubation time was 70 min at 37°C including 11 measurements with all tripeptide
concentrations (0.1 µM–3.3 mM).
Endothelin converting enzyme I (ECE-1) assay
ECE-1 activity was measured using Activity Assay Protocol by R&D systems. The assay
procedure is based on the ability of ECE-1 to degrade MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys-(DNP)OH
and the development of the fluorescence products can be measured in ex/em=320/405 nm
(Perkin Elmer Life Science, WALLAC VICTOR2 1420). The assay buffer was 0.1 mM 2-(N-morpholino)ethanesulfonic acid (MES) – 0.1 mM
NaCl –buffer (pH 6). The substrate (10 µM), the enzyme (0.005 µg) and the tripeptides
(1 µM-3.3 mM) were solved and diluted in the assay buffer. The incubation time was
60 min at room temperature including 10 measurements with all tripeptide concentrations.
Cyclooxygenase 1 (COX-1) and cyclooxygenase (COX-2) assayes
The activities of COX-1 and COX-2 were measured using commercial fluorescence-based
assay (COX Fluorescent Inhibitor Screening Assay Kit, Cayman Chemical Company, Ann
Arbor, MI, USA). The method includes 2 reactions: COX-1 and COX-2 form prostaglandin
G2 (PGG2) from arachidonic acid in the first reaction and in the second reaction PGG2 reacts with 10-acetyl-3.7-dihydroxyphenoxazine (ADPH) forming fluorescent product
resorufin which can be measured at ex/em=530/590 nm. The tripeptides (1 µM-3.3 mM)
were solved and diluted in the assay buffer. The incubation time was 60 min at room
temperature including 10 measurements with all tripeptide concentrations.
Statistical analysis
The results are presented as mean±SEM. Statistical analyses were performed using GraphPad
Prism software (version 4.02). One way analysis of variances (ANOVA) followed by Dunnett’s
multiple comparison tests were used to compare area under a curve (AUC) results. AUC
was calculated from the dose-response curves. The activities of the enzymes in different
groups were presented as percentage from the control (group without the tripeptide)
activity. Difference was considered significant if p-value <0.05.
Results
IPP, VPP and LPP inhibited POP dose-dependently at high micromolar concentrations
([Fig. 2]). IC50-values for IPP, VPP and LPP were 486±95, 761±146 and 875±192 µM (mean±SEM), respectively.
The amino acids proline and isoleucine showed no inhibitory effects on POP (data not
shown).
Fig. 2 Time-related concentration-response curves of the tripeptides for prolyl oligopeptidase
(POP) and percent AUC related to the control level. IPP (A, n=9–18), VPP (B, n= 5–12)
and LPP (C, n=3–6). One way ANOVA repeated with Dunnett’s test (*p<0.05, **p<0.01).
Interestingly, all the investigated tripeptides increased the activity of cathepsin
G ([Fig. 3]). Also amino acids proline, isoleucine and arginine elevated cathepsin G activity
([Fig. 4]). Activation was up to 50% over the control values with all the investigated tripeptides
and amino acids. Proline activated cathepsin G at lower concentrations (0.1–10 µM)
than IPP, VPP and LPP and amino acids arginine (all 1 µM-3.3 mM) and isoleucine (1–100 µM).
Fig. 3 Percent stimulatory effect of the tripeptides on cathepsin G expressed as the control
related AUC (calculated from time-concentration curves) IPP (n=3–12), VPP (n=2–3)
and LPP (n=2).One way ANOVA repeated with Dunnett’s test (*p<0.05, **p<0.01).
Fig. 4 Percent stimulatory effect of the tripeptides on cathepsin G expressed as the control
related AUC (calculated from time-concentration curves) isoleucine (n=3–8), proline
(n=6–14) and arginine (n=2–4). One way ANOVA repeated with Dunnett’s test (*p<0.05,
**p<0.01).
The tripeptides IPP and VPP showed no inhibition or activation of the other investigated
enzymes renin, chymase, NEP, ECE-1, COX-1 and COX-2 (n=3–11) at the concentrations
between 1 µM–3.3 mM (Data not shown).
Discussion
In the present study, we investigated the effects of milk casein derived tripeptides
(IPP, VPP and LPP) on other blood pressure and vascular function regulating enzymes
than ACE-1 in vitro using pure commercial enzymes. ACE-1 was left outside of the analysis
because the effect of tripeptides on ACE-1 has been already tested by us and others.
[6]
[13], Viitala R. et.al unpublished data The tested enzymes were renin, chymase, neutral
endopeptidase (NEP), cathepsin G and prolyl oligopeptidase (POP) from RAS and vascular
enzymes cyclooxygenase 1 and 2 (COX-1 and COX-2) and endothelin converting enzyme
1 (ECE-1). Renin originating from kidney cleaves angiotensinogen to Ang I which is
further converted to Ang II by ACE-1. This can also be carried out by chymase and
cathepsin G. Cathepsin G is also able to split Ang II directly from angiotensinogen.
POP forms Ang-(1–7) from Ang I and Ang II and NEP from Ang I. Also angiotensin converting
enzyme 2 (ACE-2) can create Ang-(1–7). ACE-1, NEP and POP participate in the degradation
of bradykinin into inactive fragments ([Fig. 1]) [1]
[2]
[18]
[19]
In the present study, IPP, VPP and LPP inhibited POP dose-dependently. This, however,
does not explain their blood pressure lowering effects because POP converts a vasodilatory
product Ang(1–7) from Ang I and Ang II. Ang(1–7) is able to stimulate recently describe
Mas-receptors ([Fig. 1]). Furthermore, relatively high concentrations of the tripeptides were needed for
inhibition of POP compared to ACE-1 inhibition [6]
[13]. IC50-values were higher than found in plasma after a single dose to the rat [20]. However, IPP seems to accumulate in the tissues and thus in vivo give slight pharmacological
effect.
Bradykinin is also a substrate for POP ([Fig. 1]), in addition to Ang I and Ang II at least in in vitro conditions [19]
[22]
[23]. It may be possible that Ang-(1–7), produced by POP, balances its bradykinin degradation
effects in the regulation of blood pressure and vascular function, and therefore POP
does not markedly affect blood pressure. However, POP-like activity has been shown
to be increased during ACE-1 inhibition in rats [23]. Similarly, also Ang-(1–7) levels are increased during ACE-1 inhibition in rats
and in humans [24]. Incubation of mesenteric arteries with IPP improved Ang-(1–7) – and bradykinin-induced
mesenteric artery relaxation [5]. This may be due to POP/NEP/ACE-2-Ang(1–7)-Mas-reseptor axis and due to ACE-1 inhibition
caused by IPP. It is also possible that POP and other Ang-(1–7) forming enzymes, such
as NEP and ACE-2, play a physiological role during ACE-1 inhibition or in special
pathological situations.
Despite numerous studies [18]
[19]
[21]
[22]
[24]
[25], the exact physiological role of POP is unclear. POP cleaves short naturally occurring
proline containing peptides on the carboxy side of proline residues. For that reason
it may have a role in modulating neuronal peptides and hormones containing proline
residues such as substance P and arginine vasopressin. It has been identified as a
potential target in cognitive function and in some neurodegenerative disease and POP
inhibitors have been utilized for drug development mostly for neurological disorders
and as memory enhancers [24]
[25].
Interestingly, the three tripeptides and all investigated amino acids elevated cathepsin
G activity. The results disagree with a previous study which measured the effects
of tripeptides on cathepsin G [13]. In that study, tripeptides had no effect on cathepsin G but the incubation time
was only 10 min while in the present study it was 70 min. It may be possible that
the cathepsin G activating effect was not to seen in the short incubation. This is
supported by our own in vitro vascular tests [8]. In addition, the effects of amino acids were not tested in our previous study [13]. Concerning in vivo condition amino acids isoleucine and proline stimulated cathepsin
G already at such low concentrations which could mean physiological relevance of the
findings.
Activation of cathepsin G cannot explain the decrease of blood pressure and the improvement
of vascular function because cathepsin G converts Ang II from angiotensinogen and
Ang I ([Fig. 1]). These findings support the assumption that main mechanism for the antihypertensive
effect and vascular function improvement by the tripeptides is inhibition of ACE-1.
Physiological and pathophysiological compounds can activate cathepsin G. Brecher et
al. [26] found that acetaldehyde activated cathepsin G at high millimolar concentrations
by ca. 50% similarly to the peptides and amino acids in the present study.
One limitation of the present study is that it was carried out using pure peptides
and enzymes in vitro, thus not in physiological conditions. However, these findings
show that bioactive, antihypertensive milk-derived peptides are not only ACE-1 inhibitors
but have also other effects on the components of RAS. Furthermore, the present study
indicates the complexity of the RAS with its numerous enzymes of which inhibition
or activation can change the balance between vasoconstrictive and vasodilating components
to unexpected directions. Ex vivo and in vivo experiments are warranted to clarify
possible physiological relevance of the findings
