1988 Fiscal Year Final Research Report Summary
Biochemical studies on Ca^<2+> homeostasis in phagocytic cells
| Project/Area Number |
63480380
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Research Field |
Otolaryngology
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| Research Institution | Hiroshima University (1989-1990)
Kyushu University (1988) |
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Principal Investigator |
KOGA Toshitaka Kyushu University, Faculty of Dentistry, Professor, 歯学部, 教授 (00037540)
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| Co-Investigator(Kenkyū-buntansha) |
HIRATA Masato Kyushu University, Faculty of Dentistry, Associate Professor, 歯学部, 助教授 (60136471)
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| Project Period (FY) |
1988 – 1990
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| Keywords | Phagocytic cells / Ca^<2+> ion / Inositol 1,4,5-trisphosphate / Chemotactic peptide |
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| Research Abstract |
Phagocytic cells play an important role at the sites of inflammation. The rise in intracellular Ca^<2+> is a prerequisite for exertion of the verious functions of phagocytic cells. However, little is knows about the ca^<2+> homeostasis in phagocytic cells. Here we examined this point using quinea pig peritoneal macrophages. 1. The total amount of releasable Ca^<2+> in macrophages was about 1.4 nmol/4x10^6 cells and this amount was much the same as that of the Ca^<2+> uptake by endoplasmic reticulum(ER), thereby indicating that intracellular Ca^<2+> is mainly located in ER of macrophages. The mobilized and subsequently effluxed Ca^<2+> in macrophages stimulated with chemotactic peptides (fMLP) was estimated to be 0.3 nmol/4x10^6 cells. Much the same amounts were released by inositol 1,4,5-trisphosphate(IP<@23<@D2) from ER of the cells. These results suggest that IP<@D23@>D2 may be a sole messenger for releasing Ca<@D12+@>D1 when macrophages were stimulated with fMLP. 2.To identify the p
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utative IP<@D23@>D2 receptor on ER of the macrophages an arylazide analogure of IP<@D23@>D2 was made. An azide salicyl -alanine (AS A) was iodinated with the aid of chloramine T and the product was coupled with IP<@D23@>D2 (IP<@D23@>D2-[<@D1125@>D1I]AS A) using carbonyldiimidazole as a catalyst. IP<@D23@>D2 (IP<@D23@>D2-[<@D1125@>D1I]AS A) specifically labeled three protein bands corresponding to 50K, 27K and 18KDa as possible IP<@D23@>D2 receptors on ER. 3. IP<@D23@>D2 3-kinase, which catalyzes the formation of inositol 1,3,4,5-tatrakis- phosphate was mainly located in the cytosol fraction of macrophages and was activated 2-3 fold by increasing free Ca<@D12+@>D1 concentration from 3x10<@D1-8@>D1 to 10<@D1-6@>D1M. Calmodulin (CaM) antagonists dose-dependently inhibited the Ca<@D12+@>D1-stimulated enzyme activity. The Ca<@D12+@>D1 dependence in the enzyme activity was no longer observed by reducing the contamination of CaM in enzyme fraction but it was restored by an exogenous addition of CaM. These results indicate that IP<@D23@>D2 3-kinase is a novel CaM-dependent enzyme. Less
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