| Co-Investigator(Kenkyū-buntansha) |
OSHIMA Yoshiteru Tohoku University, Graduate School of Pharmacy, Professor, 大学院・薬学研究科, 教授 (00111302)
SEYA Kazuhiko Hirosaki University, School of Medicine, Department of Pharmacology, Instructor, 医学部, 助手 (40281919)
MOTOMURA Shigeru Hirosaki University, School of Medicine, Department of Pharmacology, Professor, 医学部, 教授 (40091756)
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| Research Abstract |
1. Screening of novel bioactive substances acting on the calcium release channel.
a. ZT-B, a novel bioactive substance obtained from dinoflagellate, caused a sustained contraction of the aorta in an external Ca^<2+>-dependent manner, suggesting Ca^<2+> influx from extracellular space plays an important role in the contraction. However, it was suggested that Ca^<2+>-independent fraction of the contraction was due to the Ca^<2+> release from intracellular store sites. We are now investigating the site of action of ZT-B related to the induction of Ca^<2+> release from sarcoplasmic reticulum of smooth muscle cells.
b. Vitisin C, a novel plant oligostilbene from Vitis plants, dose-dependently inhibited the contractile responses of endothelium-intact rabbit thoracic aorta. These inhibitory effects were abolished in the presence of N^G-nitro-L-arginine methyl ester (L-NAME, 300 μM), a potent inhibitor of nitric oxide synthase. Vitisin C significantly enhanced the ^<45>Ca^<2+> influx, which was
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inhibited by nifedipine (10 μM), an L-type Ca^<2+> channel blocker. In the presence of SK&F96365, a receptor-operated Ca^<2+> channel blocker, the ^<45>Ca^<2+> influx induced by vitisin C was not affected. These results suggest that vitisin C evokes endothelium-dependent vasorelaxation through enhancing nitric oxide release, which was facilitated by Ca^<2+> influx into endothelial cells via nifedipine-sensitive Ca^<2+> channels.
2. Intracellular Ca^<2+> oscillation
PGI_2 evoked intracellular Ca^<2+> oscillation in ligament cells from patients with ossification of posterior longitudinal ligament of the spine. Some relevance of Ca^<2+> release from intracellular Ca^<2+> store site to the oscillation was suggested, because cyclopiazonic acid, a potent inhibitor for endoplasmic reticulum Ca^<2+>-pumping ATPase diminished the Ca^<2+> oscillation. Cyclic AMP is a second messenger produced by PGI_2 stimulation and an inhibitor for cAMP-dependent protein kinase markedly reduced the oscillation. The phosphorylation site would appear to be located on the regulatory protein of the Ca^<2+> release channel.
3. Expression of regulatory proteins of Ca^<2+> release channels
L6 muscle cell line can differentiates into a myotube muscle cell phenotype. To assess whether mechanical stress affects the differentiation process, cyclic stretch was applied to L6 cells attached to a deformable silicon chamber and expression of proteins related to excitation-contraction coupling was investigated. Differentiation into myotube was accelerated by cyclic stretch in L6 cells than cells in static culture. Furthermore, it has been revealed that proteins related to the regulation of Ca^<2+> release channel such as calsequestrin and triadin appeared earlier than cell in static culture. There seems to be a correlation between the appearance of these proteins and the differentiation of the contractile machinery in muscle cells including Ca^<2+> signaling system. Less
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