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
On the basis of the fi nding that Val - Tyr would be a Ca 2+ channel blocker, an ex vivo vasoconstrictive experiment using rat aorta rings was primarily con- ducted. As a result, Val - Tyr exerted a signifi cant vascular relaxation effect in SHR thoracic aorta rings in an endothelium - and ACE inhibition - independent manner (Figure 4.4 ) (Tanaka et al. 2006 ). The effec- tive and benefi cial effect of Val - Tyr in constricted aorta rings was also reported and demonstrated by Vercruysse et al. (2008) . Regarding bioactive pep- tides with respect to vascular functions, some reports to date have investigated their vascular responses.
Akpaffi ong and Taylor (1998) reported on the effect of glutathione on relaxation of SHR aorta rings, in which the tetrapeptide potentiated acetylcholine - induced relaxation in an endothelium - dependent manner. However, the vascular relaxation through improving antioxidant systems may be restrictive to peptides with antioxidant activity like glutathione, but a possibility that other peptides are also involved in endothelium - dependent relaxation can be excluded. An interesting study on vascular responses of bioactive peptides was reported on dipeptide car- nosine (beta - Ala - His) (Ririe et al. 2000 ), in which 1 mmol/L carnosine exerted ca. 10% relaxation effect in Sprague - Dawley (SD) rat aorta rings, com-
p< 0.05
100 120 140
sion l)
60 80 trictive tens % of Control
20 40 60
Const (%
0
Peptide (1 mmol/L) Control Ile-Tyr Tyr-Val Val-Tyr Val-Tyr
Figure 4.4. Vascular relaxation effect of 1 mmol/L Ile - Tyr, Tyr - Val, and Val - Tyr in 18 - week - old SHR thoracic aorta rings constricted by 30 mmol/L KCl. Val - Tyr was subjected to endo- thelium - intact and – denuded vasoconstrictive experiments.
EC: endothelium.
parable with the effect of 1 mmol/L Val - Tyr (Figure 4.4 ). The relaxation mechanism of carnosine was reported to be due to its suppression of cyclic GMP production in VSMC (Ririe et al. 2000 ), but any crucial role of carnosine in VSMC signaling path- ways was not clarifi ed. Peptides from ovalbumin, kappa - casein, egg white protein and rubisco were also reported to be a peptidic vasodilator: Ala - Asp - His - Pro - Phe from ovalbumin (Matoba et al. 1999 ), Met - Ala - Ile - Pro - Pro - Lys - Lys from kappa - casein (Miguel et al. 2007a ), and Lys - Ala - Asp - His - Pro and Tyr - Pro - Ile from egg white protein (Miguel et al.
2007b ) were clarifi ed as endothelium - dependent vasoactive peptides. Thus, it seems likely that pep- tides have an ability to regulate the vascular response, although the structural properties required for the effect remain unclear.
To clarify the structure - vasoactivity relationship of peptides, a fi rst attempt was performed using 62 synthetic di - and tripeptides in SD rat aorta rings (Tanaka et al. 2008 ). As summarized in Table 4.3 , N - terminal amino acids of tryptophanyl dipeptides would make an important contribution to vaso- relaxation action in thoracic aorta and would play an alternative crucial role in exerting a vaso- relaxation effect. Among the screened peptides, Trp - His evoked the most potent vasorelaxation effect with an EC 50 value of 3.4 mmol/L. As can be seen in Figure 4.5 , Trp - His relaxed the 50 mmol/L KCl - constricted aorta rings in an endothelium - independent manner, as Val - Tyr did. As mentioned above, carnosine had an endothelium - dependent vasorelaxation power, in which it stimulated the soluble guanylate cyclase/cyclic GMP relaxation pathways (Ririe et al. 2000 ). Arg - Ala - Asp - His - Pro from egg white proteins also acted as an endothe- lium - dependent vasodilator, through the stimulation of endothelial NO production pathways via B 1 bra- dykinin receptor (Miguel et al. 2007b ). In contrast to the endothelium - dependent vasorelaxation action of longer peptides, the endothelium - independent vasorelaxation action of Trp - His would be closely associated with some events in vascular smooth muscle layer, apart from endothelium signaling talks including the renin - angiotensin system. Interestingly, a competitive inhibition study with Ca 2+ channel
Table 4.3. Screening of vasorelaxant peptides a of 50 mmol/L KCl - constricted aortic ring. 1
Å @ Peptide a
Relaxation
(% of KCl Constriction) Å @ Peptide
Relaxation
(% of KCl Constriction)
X - Tyr X - Trp
X= Ala Å — b X= Lys Å —
Phe Å — Leu - Ile < 10
Gly Å — Val - Ile Å —
His Å — Val Å —
Ile - Phe Å — Tyr - Ile < 10
Ile - Leu Å — Trp Å —
Ile - Val Å — Trp - X
Lys Å — X= His 97
Leu - Ile Å — Val 11
Pro Å — Leu 26
Ser Å — Ala < 10
Trp - Ile Å — Gly Å —
Trp Å — Ser Å —
Tyr - Ile < 10 Met Å —
Tyr - Val Å — Glu Å —
Val - X Asp Å —
X= Phe Å — Lys Å —
His Å — Ile Å —
X - Phe Asn < 10
X= Ile Å — Arg Å —
Gly Å — Gln Å —
Pro Å — Thr
Trp < 10 Others Å —
Ala Å — Ala - Pro Å —
Val - His Å — Phe - Pro Å —
Ile - His Å — His - Ala Å —
Trp - Ile Å — His - Pro < 10
His - Ile < 10 Ile - His Å —
X - Trp Arg - Pro < 10
X= Phe Å — Phe - Ile Å —
Ile - Val < 10 Phe - His
Ile Å — Trp Å —
a Concentration of peptide was set at 5.0 mmol/L.
b No relaxation.
1 Tanaka et al. 2008 .
blockers revealed that the Trp - His induced relax- ation was not attenuated in the presence of nifedip- ine (dihydropyridine [DHP] - type Ca 2+ channel blocker). In contrast, a signifi cant attenuation of the effect was observed in the presence of verapamil (phenyl alkylamine [PAA] - type Ca 2+ channel blocker) (Figure 4.6 ). This strongly suggested that Trp - His would affect the extracellular Ca 2+ infl ux via an L - type Ca 2+ channel. Photo - affi nity analyses
demonstrated that nifedipine binds to the extracel- lular side of α 1 subunit of Ca 2+ channel protein, while verapamil favorably binds to the intracellular side (Kurokawa et al. 1997 ; Yamakage and Namiki 2002 ). Verapamil (molecular weight of 454) belongs to a PAA - type drug, having two di - methoxyphenyl groups at the end of the molecule as well as cyano group and amine in the middle. Hockerman et al.
(1997) have clarifi ed that the direct binding of basic
derived from the sardine hydrolysate relaxed the vascular constriction in a different vascular regulat- ing mechanism from Trp - His or Val - Tyr, by which endothelium heme oxygenase - 1 was activated to increase CO production, followed by the stimulation of soluble guanylyl cyclase/cyclic GMP vasorelax- ation pathway.
5. Future Prospects
So far, there are many reports regarding peptide - functionality (Hartmann and Meisel 2007 ). Some peptides showed vasorelaxation, antioxidative nitrogen of PAA to Glu from domains III and IV is
essential in eliciting effective Ca 2+ channel blocking action. This indicates that an ionization of nitrogen of PAA would be important for blocking action at the intracellular channel site. The reported pKa of verapamil was around 8.8, indicating that an ionized form of verapamil is dominant at the physiological condition (pH 7.4). According to this structural information on PAA - type blockers, the developed Ca 2+ channel blocking action of Trp - His might be associated with its structural similarity with PAA.
In a recent peptide - induced vasorelaxation study (Erdmann et al. 2006 ), it was found that Met - Tyr
ACh ACh
Wash KCl PE
PE 0.51.0 2.0
3.0 3.5
4.0 5.0 Wash