1994 Fiscal Year Final Research Report Summary
STUDIES ON THE KALLIKREIN-KININ SYSTEM : ANALYSIS USING THE GENETICALLY KININOGEN-DEFICIENT RATS
| Project/Area Number |
04454534
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | KITASATO UNIVERSITY |
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Principal Investigator |
OH-ISHI Sachiko KITASATO UNIV., SCH.PHARM.SCI., PROFESSOR, 薬学部, 教授 (70050416)
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| Co-Investigator(Kenkyū-buntansha) |
UTSUNOMIYA Iku KITASATO UNIV., SCH.PHARM.SCI., ASSISTANT PROFESSOR, 薬学部, 助手 (70168722)
YAMAKI Kohji KITASATO UNIV., SCH.PHARM.SCI., ASSISTANT PROFESSOR, 薬学部, 助手 (70174597)
HAYASHI Izumi KITASATO UNIV., SCH.PHARM.SCI., LECTURER, 薬学部, 講師 (90172999)
UENO Akinori KITASATO UNIV., SCH.PHARM.SCI., ASSOCIATE PROFESSOR, 薬学部, 助教授 (00112657)
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| Project Period (FY) |
1992 – 1994
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| Keywords | KININOGEN / KININOGEN-DEFICIENT RAT / A POINT MUTATION / SECRETION DEFECT / HIGH MOLECULAR WEIGHT KININOGEN / LLOW MOLECULAR WEIGHT KININOGEN / BRADYKININ |
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| Research Abstract |
1.Secretion defect of kininogens by the liver of B/N-Katholiek rats : Primary cultures of the hepatocytes of kininogen-deficient B/N-Katholiek and normal B/N-Kitasato rats were examined for synthesis and secretion of HMW-kininogen (HK) and LMW-Kininogen (LK) by the assessment of the incorporation of [^<35>S] -methionine. Hepatocytes of both strains of rats synthesized HK and LK similarly. Hepatocyte cultures of normal rats secreted HK and LK into the medium in 2 to 3 hrs, but cultures of kininogen-deficient rats did not secrete and retained HK and LK in the cells, which localized in the lysosomal fraction.
2.Cloning of HK cDNA of B/N-Katholiek and B/N-Kitasato rats : By an analysis of the sequence of the HK cDNA of B/N-Katholiek and B/N-Kitasato rats we found a point mutation of G to A at nucleotide 487, which locates at the heavy chain region of HK and LK.The mutation results in an amino acid transposition of alanine 163 to threonine. When a plasmid constructed with the nucleotide fragment containing G to A transition transfected into COS-1 cells , HK hardly secreted into the medium in 24 hr incubation. On the contrast, the cells transfected with the plasmide containing normal nucleotide sequence could release HK.Therefore the result suggests that a point mutation of alanine to threonine could be a cause of secretion defect of B/N-Katholiek strain.
3.Role of HK and LK in the body : By the experiment using Western blot analysis, HK molecule that had been released bradykinin, i.e. kinin-free HK,was detected in the pleural exudate of rat carrageenin pleurisy. The result indicates that at the inflammatory site kinin may be released from HK to act as an inflammatory mediator. On the other hand urinary kinin could be derived from plasma LK,because urinary kinin content in kininogen-deficient B/N-Katholiek rats increased more significantly when infused with LK than HK.
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