氏
名
論文題名
： タナッチャポーン
カムランシー
： Study on the regulation of vasocontractive Ca2+-signaling pathways by natural
compounds
（天然成分による血管収縮 Ca2+シグナル系の調節作用に関する研究）
区
分
： 甲
論
文
内
容
の
要
旨
Hypertension is a major worldwide public-health issue that causes serious cardiovascular-related
diseases. To overcome or improve hypertension disease, the preventive effect of natural compounds has been
studied by the suppression of circulatory renin-angiotensin system and/or by the improvement of local
contracted vessels. However, few studies have been conducted on the regulation of impaired vessel functions
by food compounds, since the candidates and their vasorelaxant mechanism in contracted vessels have not
been fully clarified. In the present study, thus, efforts were made to explore natural compounds possessing a
vasorelaxant ability, and their underlying mechanism was investigated on predominant vasocontractive
Ca2+-signaling pathways.
Firstly, the vasorelaxant mechanism of Trp-His that could inhibit Ca2+-calmodulin-dependent
protein kinase II (CaMK II) activity in vascular smooth muscle cells (VSMCs) was examined. A newly
developed Ca2+-encapsulation fluorescence assay using Fluo-4 probe revealed that Trp-His as well as
His-Trp inhibited the formation of Ca2+-calmodulin (CaM) complex that is a trigger for CaMK II activation.
The structural requirement for such basic dipeptides to exert the action was a peptide skeleton, irrespective
of the sequence. In addition, an imino proton in the imidazole ring of His, and the indole moiety of Trp
residue were involved in the inhibition of Ca2+-CaM complex formation. In order to get information on the
interaction of basic peptide with CaM, in silico analysis was conducted to simulate the complex in virtual
water system. After optimizing the stable conformations of dipeptides and CaM in water, molecular
dynamics (MD) docking experiments were performed with a SYBYL/Surflex-dock program, and an MD
simulation by AMBER was run for 2500 ps. As a result, Trp-His and His-Trp interacted with Ca2+-binding
sites of CaM at Asp22 and Glu31 by hydrogen bonds with binding free energy of − 49.1 and − 68.0 kJ/mol,
respectively. These findings strongly demonstrated that Trp-His competed with free Ca2+ to bind to CaM,
resulting in the inhibition of Ca2+-CaM complex formation, leading to the suppression of vasocontractive
Ca2+-signaling pathways via Ca2+-CaM/CaMK II/voltage-dependent L-type Ca2+ channel (VDCC)
phosphorylation in VSMCs.
Secondary, an attempt was made to identify natural peptides possessing CaMK II inhibitory activity,
as similar to Trp-His and His-Trp. By the hydrolysis of wheat germ proteins by thermolysin two
Trp-containing dipeptides among eight identified peptides in the hydrolysate were found to inhibit CaMK II
activity. Both peptides were unlikely to bind to CaM in the Ca2+-binding sites, but inhibited the activation of
CaMK II directly. Finally, an investigation on regulatory action of natural nucleotides on vasocontractive
Ca2+-signaling pathways was performed, in which nucleotides in Candida utilis yeast extract have an ability
to regulate Ca2+-signaling pathways, as similar to basic dipeptides.
In conclusion, the present study demonstrated that natural compounds had a potency to regulate
impaired vessel functions through the regulation of CaM-related Ca2+-signaling pathways. This study may be
of great value to get insights of natural compounds into preventive effect on vascular-related diseases.