Mitsuru Tanaka

Antiproliferative action of an angiotensin I-converting enzyme inhibitory peptide, Val-Tyr, via an L-type Ca2+ channel inhibition in cultured vascular smooth muscle cells.

Recent antihypertensive studies have demonstrated that small peptides with angiotensin I-converting enzyme (ACE) inhibitory activity had an ability to lower or to modulate a pressor blood pressure response in mild hypertensive subjects. However, the underlying mechanisms still remain unclear. Based on our previous finding that a small peptide, Val-Tyr (VY), was accumulated in the rat aorta and kidney as well as in the circulating blood system, we here investigated whether antihypertensive small peptides exert an antiproliferative effect on serum- or mitogen-induced human vascular smooth muscle cells (VSMCs). Treatment with some ACE inhibitory small peptides (VY, Ile-Trp [IW], and Ile-Val-Tyr [IVY]) had diverse effects on serum-stimulated VSMC proliferation that were independent of their ACE inhibitory activity, though only VY exerted a potent antiproliferative action. VY also showed a greater inhibition of WST-8 incorporation in response to angiotensin (Ang) II-stimulation than the other two small peptides. The attenuation of Ang II-stimulated WST-8 incorporation by VY was not affected by Ang II receptor antagonists (losartan and saralasin ([Sar1, Ile8]-Ang II)), indicating that the antiproliferative action of VY may not be due to the peptides antagonistic effect against Ang II receptors. Treatment with VY had a significant inhibitory effect on the WST-8 incorporation induced by the stimulation of a voltage-gated L-type Ca2+ channel agonist, Bay K 8644. Even in the presence of a K+ channel blocker (paxillin) the inhibition was apparent, suggesting that VY inhibited the proliferation of VSMCs by serving as a natural L-type Ca2+ channel blocker, but not as a K+ channel agonist.

Endothelium-independent vasodilation effect of di- and tri-peptides in thoracic aorta of Sprague-Dawley rats.

The goal of this study was to elucidate the structure-activity relationship for vasodilating peptides and their underlying mechanism. In this study, we synthesized 62 di- and tri-peptides having aromatic amino acid residues (Tyr, Trp and Phe). Among them, only 4 peptides (HW, WH, WL and WV) evoked an apparent vasodilating effect in 50 mM KCl-contracted aortic rings in the descending order of WH>HW>WL>WV; WH showed the vasodilating activity with an EC50 of 3.4 mM. Within our experimental results, it seems likely that Trp residue at the N-terminal would play a role in eliciting vasodilating effect. No appearance of vasodilating effect for stereoisomers of WH with D-configuration revealed that the vessel would recognize the L-configuration of WH. The presence of angiotensin I-converting enzyme (ACE) inhibitor (50 nM enalaprilat) did not affect the WH-induced vasodilating effect, though WH showed a slight ACE inhibitory activity (IC50: 93 microM). The effect was also observed in the endothelium-denuded aortic rings. In contrast, WH provoked a significant displacement to the right in the vasodilating curve in the presence of 30 nM verapamil, while no shift was observed in the presence of 2.5 nM nifedipine. These results indicate that WH, a vasodilating di-peptide, would exert a vasodilation via the suppression of Ca2+ influx into KCl-induced depolarized aortic rings. The present study also suggests that the binding site of WH to the Ca2+ channel may be competitive to that of phenylalkylamine-type voltage-gated L-type Ca2+ channel blocker.

Vasodilating dipeptide Trp-His can prevent atherosclerosis in apo E-deficient mice.

Most of the investigations for an alternative medicinal treatment on atherosclerosis have been focused on natural or dietary compounds including phytochemicals. So far, few studies regarding anti-atherosclerotic small peptides except for tetrapeptide of Lys-Arg-Glu-Ser have been reported. The present study was, thus, to investigate whether dipeptide Trp-His, which is one of vasodilating small peptides, could reduce atherosclerotic lesions in apo E-deficient mice fed a high-fat diet. The animal study involved a 9-week-successive administration of Trp-His at a dose of 0, 10 or 100 mg/kg per d. After 9-week administration, en face analyses provided the first direct evidence that the atherosclerotic lesion area was significantly reduced by 27 and 38 % for Trp-His dosed at 10 and 100 mg/kg per d, respectively, compared with the control group. Administration of Trp-His did not affect growth parameters such as body weight and feeding efficiency (P>0.1). Total serum cholesterol and HDL-cholesterol as well as lipid profiles in the liver did not differ between the tested groups. Taken together, the anti-atherosclerotic effect of dipeptide Trp-His should be addressed into physiological functions of bioactive peptides, in which the dipeptide may elicit the power by alternative mechanism(s), not by the regulation of lipid metabolism.

Trp-His, a vasorelaxant di-peptide, can inhibit extracellular Ca2+ entry to rat vascular smooth muscle cells through blockade of dihydropyridine-like L-type Ca2+ channels

Our previous findings regarding the biological activities of small peptides revealed that a di-peptide, Trp-His (WH), could play a role in the prevention of vascular lesions, including cell proliferation and atherosclerosis. Its vasoprotective effects could be associated with suppression of the vasocontraction signaling cascade, but the underlying mechanism(s) remains obscure. In this study, we attempted to elucidate the vasoprotective mechanism of WH, in opposing the proliferation of rat vascular smooth muscle cells (VSMCs). In VSMCs from 8 week-old male Wistar rat thoracic aortae, WH evoked a significant dose-dependent anti-proliferation effect, without cytotoxicity. In mitogen-stimulated cell experiments, 300 μM WH inhibited cytosolic Ca(2+) elevation in VSMCs induced by 10 μM angiotensin II (Ang II). Furthermore, WH suppressed extracellular Ca(2+) entry into CaCl(2)-stimulated VSMCs. The biological capacity of WH as an intracellular Ca(2+) ([Ca(2+)](i)) suppressor was also proven when 50 μM Bay K8644 was used to enhance Ca(2+) entry via a voltage-dependent l-type Ca(2+) channel (VDCC) and 300 μM WH elicited a 23% reduction in [Ca(2+)](i). The absence of a reduction of the [Ca(2+)](i) by the mixture of tryptophan and histidine revealed the importance of the peptide backbone in the [Ca(2+)](i) reduction effect. Furthermore, the WH-induced [Ca(2+)](i) reduction was abolished by verapamil, but not by nifedipine, indicating that WH likely binds to an extracellular site of the VDCC at a site similar to that of the dihydropyridine type-Ca(2+) channel blockers.

Theaflavins enhance intestinal barrier of Caco-2 Cell monolayers through the expression of AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1

We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (Papp) of fluorescein in Caco-2 cells pretreated with 20 μM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any Papp reduction, purpurogallin pretreatment resulted in a significant Papp reduction similar to that of TF-3′-O-gallate (TF3′G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the Papp reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3′-O-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3′G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells. Graphical Abstract Theaflavins can close the tight junction or improve the intestinal membrane barrier via AMPK activation in Caco-2 cells.

Endothelium-dependent vasorelaxation effect of rutin-free tartary buckwheat extract in isolated rat thoracic aorta

The aim of this study was to point out the potential of tartary buckwheat on vascular functions. A nonabsorbed fraction of hot-water extract of tartary buckwheat on a SP70 column (TBSP-T), which was free from rutin, was used for this aim. In a contractile experiment using Sprague-Dawley rat thoracic aorta rings contracted by 1.0 microM phenylephrine (PE) or 50 mM KCl, TBSP-T evoked a significant vasorelaxation [EC50 (mg/ml): PE; 2.2; KCl, 1.9]. By a further fractionation of TBSP-T by liquid-liquid partitioning into basic, neutral and acidic fractions, a marked enhancement of vasorelaxation effect was observed only for acidic fraction (EC50, 0.25 mg/ml). The action of acidic fraction was significantly attenuated in endothelium-denuded aortic rings and in the presence of nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM). The fraction also enhanced the cyclic guanosine monophosphate (cGMP) production in aortic rings contracted with PE [cGMP (pmol/mg protein): PE, 7.2+/-2.3; PE+Acidic fraction, 35+/-8]. These results indicate that acidic fraction could mediate NO/cGMP pathways, thereby exerting endothelium-dependent vasorelaxation action. In conclusion, tartary buckwheat was proven to regulate vascular tones and have latent acidic candidates except for rutin.

Vasodilating effect of di-peptides in thoracic aortas from spontaneously hypertensive rats.

In this study, we found that antihypertensive di-peptide Val-Tyr (VY) showed a vascular relaxation effect in KCl-induced contraction of thoracic aorta rings from 18-week-old spontaneously hypertensive rats among di-peptides of VY, Ile-Tyr, and Tyr-Val irrespective of their angiotensin I-converting enzyme inhibitory activity. The effect was endothelium-independent, and was closely associated with vascular responses in the vascular smooth muscle layer.

His-Arg-Trp potently attenuates contracted tension of thoracic aorta of Sprague-Dawley rats through the suppression of extracellular Ca2+ influx

In the present study, we primarily attempted to identify di- and tri-peptides showing potent vasodilation in 1.0 microM phenylephrine-contracted thoracic aortas of Sprague-Dawley rats. Synthetic 15 Trp-His (WH) skeleton analogues were used for rat aorta rings force measurements, since WH was found to be a vasoactive di-peptide so far. Among the synthesized peptides consisted of both His and Trp amino acid residues, His-Arg-Trp (HRW) was found to evoke the most potent vasodilation with an EC50 value of 1.2+/-0.08 mM in an endothelium-independent manner, while no effect was evoked by a mixture of individual amino acids. In addition to the structure of tri-peptides-activity relationship, chemically modified HRW analogues, i.e., 1- or 3-methyl-His-Arg-Trp and His-citrulline-Trp demonstrated the structural importance of tri-peptide to evoke the vasoactivity as following factors: (1) Neutral imidazole and indole groups from His and Trp residues at N- and C-terminals, respectively and (2) basic amino acids at the middle position. In mitogen (10 microM angiotensin II or 50 microM Bay K8644)-stimulated vascular smooth muscle cells, vasoactive HRW (100 microM) caused significant [Ca(2+)](i) reduction to an extent of >30%. Thus, our results suggest that HRW caused vasodilation action via an endothelium-independent mechanism which probably involves the suppression of extracellular Ca2+ influx through voltage-gated l-type Ca2+ channel.

Highly-Sensitive Detection of Free Advanced Glycation End-Products by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry with 2,4,6-Trinitrobenzene Sulfonate Derivatization

The common derivatization method of primary amino groups by 2,4,6-trinitrobenzene sulfonate (TNBS) was applied to the detection of free adducts of advanced glycation end-products (AGEs), in combination with liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM). The proposed TNBS-MS method provided a surprisingly significant improvement of the detection of AGE-free adducts (e.g., by a factor of >1000 for methylglyoxal-derived hydroimidazolone) with a detection limit of 1.0 nM (10 fmol/injection volume), which was due to the high ionization efficiency of the derived trinitrophenol moiety and its hydrophobicity. With the aid of stable-isotope-labeled internal standard (MG-H1-d3), the convenient TNBS method that allowed the derivation of AGE-free adducts bearing amino groups under mild reaction conditions (30 mM, pH 8.5, 30 min, 30 °C) also permitted successive detection and quantification of five typical AGE-free adducts at nanomolar levels in rat plasma (50 μL) with high reproducibility (2-9% of RSD) and recovery (93-113%), using LC-MS/MS-MRM.

Ferulic acid enhances the vasorelaxant effect of epigallocatechin gallate in tumor necrosis factor-alpha-induced inflammatory rat aorta

Previously, we demonstrated synergistic enhancement of vasorelaxation by combination treatment with Trp-His and epigallocatechin gallate (EGCg) in intact rat aorta. The aim of the present study was to determine whether this vasorelaxant synergy could be recapitulated in tumor necrosis factor-alpha (TNF-α)-induced inflammatory rat aorta, and to determine the extent of its modulation by anti-inflammatory phenolic acids. Synergistic enhancement of vasorelaxation in rat aorta by Trp-His and EGCg was significantly attenuated in the presence of TNF-α, an effect that was reversed by the addition of ferulic acid (FA, 250 μM). Moreover, FA markedly enhanced EGCg-induced vasorelaxation, but not Trp-His-induced vasorelaxation, in TNF-α-treated aorta. Structure-activity analysis showed that the unsaturated 2-propenoic moiety and the methoxy group of FA were important for the enhancement of vasorelaxation by EGCg. The stimulation of EGCg-induced vasorelaxation by FA was antagonized by the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine acetate, while FA enhanced vasorelaxant properties of the endothelial nitric oxide (NO) synthase activator acetylcholine in TNF-α-treated inflammatory aorta. Moreover, the EGCg-stimulated NO production was also enhanced by FA in TNF-α-treated aorta. These data indicate that stimulation of NO production by FA enhances the vasorelaxant properties of EGCg in TNF-α-induced inflammatory aorta.