| Co-Investigator(Kenkyū-buntansha) |
MAEDA Sadaaki Osaka University Faculty of Dentistry, Research Instructor, 歯学部, 助手 (00135732)
YONEHARA Norifumi Osaka University Faculty of Dentistry, Research Instructor, 歯学部, 助手 (70124534)
SAITO Kihachi Osaka University Faculty of Dentistry, Assoc. Prof., 歯学部, 助教授 (40110788)
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| Budget Amount *help |
¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1991: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1990: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1989: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Research Abstract |
The present study was aimed to investigate the mechanism of enkephalin (EK) production induced b. y bradykinin (BK) in dental pulp of the rat, using subcellular fractions or cultured fibroblasts of the rat incisor pulp tissue. In experiments using subcellular fractions, it was found that (1) the en2yme activity of EK production was localized in lysosomal fraction and (2) EK precursor proteins were conatined in microsomal and supernatant fractions. Thereafter, EK producing enzyme was isolated from the lysosomal fraction and purified. (3) lt was demonstrated that the en2yme was a cysteine proteinase, cathepsin B (M. W. ; 23, 600). On the other hand, EK precursor protein was isolated from the microsomal and supernatant fractions. and purified. (4) lt was certified that a common protein (M. W. ; 58, 000) in both fractions was a major precursor protein. (5) The purified cathepsin B was not activated by BK but the enzyme in entire lysosomal structure was activated by it. In experiments using
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pulpal cells, fibroblasts were easily cultured from the pulp tissue. (6) lt was immunocytochemically or immunochemically demonstrated that the cells contained EKs and their precursor protein. (7) lncubation of the cells with BK, des-Arg9-BK, or arginine (Arg) increased cathepsin B activity of the cells, thereby EKs were produced. (8) The actions of these agents was dependent on concentration of intra- and extracelluar Ca^<++>. In experiments using the fibroblasts with various enzyme inhibitors and antagonist, (9) it was found that the actions of BK and des-Arg^9-BK were mediated through B_1-receptor coupled with an islet-activating protein (IAP)-sensitive GTP-binding protein (G-protein), followed by activation of phospholipase C (PLC), a key en2yme in cathepsin B activation. The activation of PLC induced an elevation of intracellular Ca^<++> concentration, followed by activation of various protein kinases, such as protein kinase (PK) C, Ca^<++>/calmodulin-dependent PK and CGMPdependent PK. It was also found that (10) IAP-sensitive G-protein was not involved in activation of cathepsin B by Arg, but Arg was uptaken into the cells to form kyotorphin (KTP), an EK releaser, which acted on KTP receptor of cell membrane, resulting in activation of cathepsin B, and in addition, to form nitric oxide (NO), an activator of guanylate cyclase, resulting in production of CGMP. In conclusion, it was considered that BK action involved in cathepsin B activation of the fibroblasts was due to a combination effect of des-Arg^9-BK and Arg which were produced by an enzymatic degradation of BK by which various protein phosphorylation steps were induced. Less
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