Toshiro Matsui and Mitsuru Tanaka
Contents
1. Introduction, 43
2. Hypertension and the Renin - Angiotensin System, 44
3. Design of ACE Inhibitory Peptides, 44 4. Antihypertensive Mechanism of Small
Peptides, 46
4.1. Inhibition of the Renin - Angiotensin System by ACE Inhibitory Peptides, 46
4.2. Regulation of Vascular Events by Dipeptides, 47
4.3. Relaxation of Vascular Constrictive Events by Dipeptides, 48
5. Future Prospects, 51 6. References, 53
1. Introduction
Peptides, which are condensed amino acids, have been well documented as physiologically functional compounds in nature. In this chapter, we review their updated antihypertensive mechanism as well as the development of antihypertensive food products, because small peptides including di - and tripeptides are favorably absorbed through intestinal peptide transporter PepT1.
In Japan, the number of hypertensives and high - normal hypertensives (systolic blood pressure [SBP] > 135 mmHg and/or diastolic blood pressure [DBP] > 85 mmHg) was ca. 55 million in 2006 (Japanese Ministry of Health, Labour and Welfare).
In the case of essential hypertension, which affl icts
> 90% of all hypertensions, the onset followed by
cardiovascular disease, renal dysfunction, or periph- eral vascular disease is closely associated with life - and/or food - style, including excess high - salt intake (Kawano et al. 2007 ). Namely, disruption of Na + - K + balance or increase in fl uid volume as well as increasing vascular resistance will lead to promotion of blood pressure (BP). The most benefi cial improve- ment for essential/borderline hypertension is, there- fore, said to be one that achieves modifi cation of food - style and/or moderate exercise. In addition to such lifestyle modifi cation treatments, peptide research has become one of the growing fi elds for preventing - medicinal chemistry, since some clinical evidence shows the effi ciency of peptide intake for improving hypertension disease. In Japan, evidence - based foods with health claim (FOSHU, or foods for specifi ed health use) have been developed and accepted by the Japanese Ministry of Health, Labour and Welfare ( http://www/mhlw.go.jp/ ). For FOSHU products effective for modulating BP in borderline hypertensives, seven products (tryptic hydrolysate of casein, Katsuobushi [dried bonito] oligopeptide,
Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals Edited by Yoshinori Mine, Eunice Li-Chan, and Bo Jiang
© 2010 Blackwell Publishing Ltd. and Institute of Food Technologists ISBN: 978-0-813-81311-0
dase) to potent vasoconstrictor Ang II by cleaving the dipeptide His - Leu from the C - terminal of Ang I. The major physiological role of Ang II is to exert a vasoconstrictive effect at the vessel wall via the binding of Ang II to Ang II receptor (AT1) (Millatta et al. 1999 ). Ang II also participates in the increment of extracellular fl uid volume through a stimulation of adrenal aldosterone release, by which renal tubular sodium reabsorption is enhanced. Thus, it has been recognized that the circulatory renin - angiotensin system plays a crucial role in the devel- opment and maintenance of hypertension. Updated research (Bader and Ganten 2008 ) also helps us to understand the importance of the local renin - angio- tensin system in regulating BP promotion, where new candidates of (pro)renin and ACE2, an Ang (1 - 7) producing enzyme, involve the local (kidney, heart, vessel, or brain) BP regulation system like autocrine/paracrine hormone (Figure 4.1 ).
3. Design of ACE Inhibitory Peptides In order to prevent the pathogenesis of hypertension or to treat an elevated BP, suppression of Ang II production via inhibition of ACE activity would be of great benefi t, because both renin - angiotensin and kinin - kallikrein systems involve ACE action. In addition, taking into consideration that ACE has four functional amino acid residues of Tyr, Arg, Glu, and Lys at the active site, and three hydrophobic aqueous extract from Mycoleptodonoide s ait-
chisonii , Lactobacillus helveticus - fermented milk [sour milk], sardine peptide, seaweed peptides, and sesame peptides) are available in the local market, most of which involve the inhibition of angiotensin I – converting enzyme (ACE) by small peptides (Table 4.1 ). The effi cacy of peptide intake for bor- derline hypertensives is evidentially developed on the basis of extensive intervention trials, but such an effective FOSHU medication led us to a potential question of whether the antihypertensive effect of peptides is achieved only by ACE inhibition or sup- pression of the renin - angiotensin system, like thera- peutic ACE inhibitory drugs.
2. Hypertension and
the Renin - Angiotensin System
Arterial BP regulation is mainly achieved by diverse metabolic systems: pressor and depressor hormonal systems and nerve systems (Sealey and Laragh 1990 ). Among these, the renin - angiotensin ( - aldo- sterone) system is thought to be one of the predomi- nant pressor systems, widely occurring in not only the circulatory blood system but also in diverse organs such as brain, lung, aorta, and kidney. In this system, angiotensinogen from the liver is primarily cleaved by renal renin to produce angiotensin (Ang) I that is a decapeptide. Ang I is then converted by the action of ACE (EC 3.4.15.1, dicarboxypepti-
Table 4.1. Antihypertensive FOSHU products.
Functional Ingredient Active Peptide Daily Intake
Reduc - tion of
SBP/DBP a (mmHg) Reference Tryptic hydrolysate of casein Phe - Phe - Val - Ala - Pro - Phe -
Pro - Glu - Val - Phe - Gly - Lys
20 g 4.6/6.6 Sekiya et al. 1992
Katsuobushi (dried bonito) oligopeptide
Leu - Lys - Pro - Asn - Met Leu - Lys - Pro
1.5 g 11.7/6.9 Fujita et al. 2001 Aqueous extract from
Mycoleptodo - noides aitchisonii
Ile - Tyr 1 g 9.4/6.7 Tsuchida et al. 2001
Lactobacillus helveticus — fer - mented milk (sour milk)
Val - Pro - Pro Ile - Pro - Pro
VPP equiv. : 2.53 mg IPP equiv. : 1.52 mg
12.5/6.2 Kajimoto et al. 2001
Sardine peptide Val - Tyr 4 g 9.3/5.2 Kawasaki et al. 2000
a SBP: systolic blood pressure; DBP: diastolic blood pressure.
of 0.018 mg - protein/mL. After a 4 - week admini- stration of the sardine hydrolysate (4 g/day) to 29 borderline hypertensive volunteers (systolic BP/
diastolic BP, peptide - drink: 146.4/90.5 mmHg;
placebo: 145.5/92.3 mmHg), a signifi cant BP reduc- tion (systolic BP/diastolic BP: 9.3/5.2 mmHg) was observed in the peptide - drink group, whereas there was no BP change in the placebo group (Kawasaki et al. 2000 ). The human volunteer study provided some useful information on the BP - lowering effect of natural antihypertensive food: no rebound phe- nomena and no side effects, such as dry cough, which are often observed when ACE inhibitory drugs are taken. Therefore, these fi ndings eviden- tially reveal that the intake of antihypertensive FOSHU products prepared from natural food resources is of great benefi t for regulating or improv- ing an elevated BP. It seems likely that FOSHU products containing ACE inhibitory peptides possess a similar extent of BP - lowering power (Table 4.1 ).
Contrary to the prevalence, there was a substan- tially great difference in ACE inhibitory activity between therapeutic drug (e.g., captopril: IC 50 = 0.021 μ mol/L) and peptides (e.g., Leu - Arg - Pro:
IC 50 , 0.27 μ mol/L). Some reports (FitzGerald et al.
binding subsites, favorable blockade of ACE action would be achieved by small peptides or peptidic inhibitors having high affi nity with active sites. The fi nding that the transporter - recognized di - and tri- peptide length is expressed at the intestine (PepT1) (Minami et al. 1992 ; Vig et al. 2006 ) also allows us to investigate the possible functionality of small pep- tides after absorption. From this point of view, many ACE inhibitory peptides have been designed and identifi ed from natural proteins (Matsui and Matsumoto 2006 ); as a therapeutic ACE inhibitory drug, captopril or enalapril, was designed on the basis of a basal structure of Ala - Pro or Phe - Ala - Pro, respectively (Hooper 1991 ). This is the reason why small peptides are targeted for developing antihypertensive food through ACE inhibitory action. ACE inhibitory peptides ( > 400) reported so far provide the evidence that small peptides with hydrophobic and aromatic amino acid residues such as Tyr, Phe, Trp, and Pro at the C - terminal have a potent ability to inhibit ACE activity with an IC 50 value of < 100 μ mol/L (Matsui and Matsumoto 2006 ). A successful human study was reported using sardine muscle hydrolysate by alkaline prote- ase showing an ACE inhibitory power of IC 50 value
Renin-Angiotensin System (RAS) A i t i
Prorenin Angiotensinogen
Renin
Renin (Pro)renin receptor Angiotensin I
ACE ACE Angiotensin(1-9)
ACE
ACE ACE2ACE2
Angiotensin(1-7) Angiotensin II ACE2
ACE2
Mas-R AT2-R AT1-R
Hypertensive events Antihypertensive events
Figure 4.1. Updated circulatory renin - angiotensin system.
hours (systolic BP9h: 120.7 ± 2.3 mmHg) (Matsui et al. 2003 ). This preferable result demonstrates that the antihypertensive effect induced by ACE inhibi- tory peptides closely correlates with human renin - angiotensin system. In contrast to these fi ndings, there was no difference in plasma ACE activity between control and sample groups at 6 hours, while a transient inhibition was observed at 1 hour after the administration. A transient ACE inhibition of Val - Tyr was also observed in spontaneously hyper- tensive rats (SHR) and human studies (Kawasaki et al. 2000 ; Matsui et al. 2004 ). For ACE activity at diverse organs, a long - term suppression of ACE activity was observed in the kidney and aorta, while no change was observed in plasma ACE activity (Figure 4.2 ).
These results suggested that both organs were targeting tissues of Val - Tyr. In other words, increas- ing plasma dipeptide has the power to suppress the pressor Ang II production via ACE inhibitory action transiently, but an alternative antihyperten- sive action occurring at the local tissues should be involved in the long - lasting BP - lowering effect of dipeptide. A similar long - lasting BP - lowering effect of ACE inhibitors was observed in an acute SHR administration study of spirapril as an ACE inhibi- tory drug (Okunishi et al. 1991 ) and 5 - caffeoylquinic acid as an acetylcholine receptor mediator (Suzuki et al. 2002 ). A prolonged effect of the latter was reported to be due to slow metabolism of it to the candidate compound, ferulic acid, in the circulatory system. On the contrary, the effect induced by spi- rapril was reported to result from its long - lasting ACE inhibition at the aorta. Additionally, the fi nding that a young THM had already developed hyperten- sion partly due to a remarkable vascular hypertrophy by excess vascular Ang II production led us to spec- ulation that Val - Tyr or antihypertensive peptide might suppress an enhanced vascular renin - angio- tensin system. The fi ndings that (1) plasma Val - Tyr level at 1 hour was as little as in normotensives (12 mg dose of Val - Tyr: Cmax; 1.9 ± 0.1 pmol/mL - plasma) (Matsui et al. 2002 ), much lower than that of captopril (Cmax at 25 mg dose; 603 pmol/mL - plasma) (Jankowski et al. 1995 ), and (2) in vitro ACE inhibitory activity of Val - Tyr (IC 50 ; 26 μ mol/L) 2004 ) have also revealed the controversies between
in vivo antihypertensive effect and in vitro ACE inhibitory activity of peptides. As summarized in Table 4.2 , there seems to be no relationship between the BP - lowering effect of a given ACE inhibitory peptide and its IC 50 value. In this regard, it remains unclear whether the antihypertensive effect of ACE inhibitory peptides is elicited only due to ACE inhi- bition; and, as can be seen in the later sections, we will fi nd the potential involvement of small peptides in local BP regulation systems.
4. Antihypertensive Mechanism of Small Peptides
4.1. Inhibition of the Renin - Angiotensin System by ACE Inhibitory Peptides
Genetically defi ned Tsukuba - Hypertensive Mouse (THM) was used to demonstrate the antihyperten- sive mechanism of small peptides, in which the pathogenesis of hypertension is restrictively deter- mined by an enhanced renin - angiotensin system induced by human renin and angiotensinogen genes.
Therefore, a BP lowering of THM would refer to a suppression of enhanced human renin - angiotensin system. As a result, a single oral administration of Val - Tyr, a predominant ACE inhibitor in sardine hydrolysate to 11 weeks THM (0.1 mg/g), was proven to cause a long - lasting BP reduction up to 9
Table 4.2. Reported antihypertensive peptides from natural proteins in spontaneously hypertensive rats.
Peptide Source
IC 50 a ( μ mol/L)
Decrease in SBP (mmHg) / dose (mg/kg) Tyr - Pro α s1 - casein 720 − 32.1/10 1 Leu - Lys - Pro Dried bonito 0.32 − 16/9 2 Ile - Pro - Pro β - casein 5 − 28.3/0.3 3 Val - Tyr Sardine muscle 26 − 43.4/10 4 a Concentration of inhibitor required to inhibit 50% of the ACE activity.
1 Yamamoto et al. 1999 . 2 Fujita and Yoshikawa 1999 . 3 Nakamura et al. 1995 . 4 Matsui et al. 2004 .
Ile - Trp (IC 50 ; 2.0 μ mol/L), and Ile - Val - Tyr (IC 50 ; 0.48 μ mol/L), Val - Tyr was found to show a marked antiproliferation action in serum - stimulated VSMC growth (46% decrease in the cell number at 1 mmol/L Val - Tyr) (Matsui et al. 2005 ). Although Ile - Val - Tyr also caused a slight decrease in the growth to 82%
of control, Ile - Trp was no longer an antiproliferative peptide, being provided a confl icting result with its ACE inhibitory potentials.
Taking into consideration the signal - transduction pathway via AT 1 - receptor, an Ang II stimulation experiment was then conducted. In the presence of the peptides at a concentration of 1 mmol/L, a potent suppression of the WST - 8 incorporation into Ang II – stimulated VSMC for Val - Tyr was observed, with a reduction to ca. 65% of the control (Figure 4.3 ). Captopril as well as Ile - Trp and Ile - Val - Tyr did not show any infl uence on the incorporation was much weaker than that of captopril (IC 50 ;
0.021 μmol/L), also suggest little contribution of plasma Val - Tyr increment to long - lasting BP - lowering effect.
4.2. Regulation of Vascular Events by Dipeptides
Antihypertensive peptide may play a potential role in regulating the vascular function, though the mech- anism at the aorta remains unclear. Thus, our next trial was to demonstrate the latent function in the cell line experiment. Human vascular smooth muscle cell (VSMC) was used, since the aorta was reportedly one of the accumulated tissues of some ACE inhibi- tory peptides and directly responsible for vasocon- striction tone. In a VSMC proliferation experiment in 5% FBS - SmBM using Val - Tyr (IC 50 ; 26 μ mol/L),
% Reduction of SBP
Plasma
40 50
ity sma)
0 10
20 30
ACE activ (mU/mL plas
-10
0 10
0 1 6
Time (h)
# #
-20
Kidney
5
Aorta
5
# #
# # # #
0 1 3 6 9
2 3 4
ACE activity U/mg protein) 2
3 4
ACE activity U/mg protein)
＊＊ ＊＊
Time after administration (h)
0 1
0 1 6
A (m
＊ ＊
0 1
0 1 6
A (mU
Time (h) Time (h)
Figure 4.2. Change in systolic blood pressure (A) and ACE activities (B) of 18 - week - old spontaneously hypertensive rats after a single oral administration of 10 mg/kg Val - Tyr ( 䊉 ) or control ( 䊊 ). Each value is expressed as mean ± SEM (n = 5). ## p < 0.01 compared with the control group. * p < 0.05, * * p < 0.01 compared with 0 hours.
on bioactive small peptides responsible for blocking voltage - gated L - type Ca 2+ channel.
4.3. Relaxation of Vascular Constrictive Events by Dipeptides
Either vascular constriction or relaxation closely relates with VSMC actions, which are critical for hypertension disease including vascular or arterio- sclerotic lesions (Dzau 2001 ). VSMC proliferation, hypertrophy, or migration is induced by Ang II and/
or norepinepherine stimulation; among these Ang II that acts as an autocrine/paracrine mediator at the renin - angiotensin system plays a prominent role in the pathogenesis of vascular lesions via cell proliferation (Mehta and Griendling 2007 ). Thus, the application of dipeptides having voltage - gated L - type Ca 2+ channel blocking action, like AT 1 - receptor antagonist (Miura et al. 2003 ) or Ca 2+
channel blocker (Stepien et al. 1997 ), for the treat- ment of vascular lesion - related diseases including hypertension would be highly appreciated.
at a concentration of 1 μmol/L, which strongly suggested that the antiproliferative effect induced by Val - Tyr was not a result of aortic ACE inhibition.
A similar suppression effect of the incorporation by Val - Tyr was observed, irrespective of the presence of AT 1- receptor selective antagonist (losartan) or nonselective AT - receptor antagonist (saralasin) (see Matsui et al. 2005 , indicating that Val - Tyr had no antagonistic effect against Ang II – related recep- tors). When Bay K 8644 acting as a mitogen through voltage - gated L - type Ca 2+ channel stimulation was used, 1 mmol/L Val - Tyr signifi cantly inhibited the increasing incorporation stimulated by 1 μ mol/L Bay K 8644, like that which 1 μ mol/L verapamil (therapeutic L - type Ca 2+ channel blocker) inhibited (Figure 4.3 ). In contrast, the presence of paxillin (K + channel blocker) did not affect the inhibition by Val - Tyr. This provided evidence that the antiprolif- eration by Val - Tyr would be in part due to inhibition of extracellular Ca 2+ infl ux into VSMC by blocking voltage - gated L - type Ca 2+ channel, not by stimulat- ing K + channel. So far, no study has been reported