STUDIES ON THE KALLIKREIN-KININ SYSTEM : ANALYSIS USING THE GENETICALLY KININOGEN-DEFICIENT RATS

1.B/N-Katholiek大鼠肝脏激肽原分泌缺陷：原代培养的B/N-Katholiek大鼠肝细胞和正常B/N-Kitasato大鼠肝细胞合成和分泌高分子激肽原(HK)和低分子激肽原(LK)，通过[^&lt；35&gt；S]-蛋氨酸掺入法检测。两种品系大鼠肝细胞合成HK和LK相似。2.B/N-Katholiek和B/N-Kitasato大鼠HK基因的克隆：通过对B/N-Katholiek和B/N-Kitasato大鼠HK基因序列的分析，我们发现487位核苷酸G到A的点突变，该突变位于HK和LK的重链区域。将含有G-A转换的核苷酸片段构建的质粒导入COS-1细胞，在24小时内，HK几乎不能分泌到培养液中。结果提示，丙氨酸到苏氨酸的点突变可能是B/N-Katholiek菌株分泌缺陷的原因之一。3.HK和LK在体内的作用：通过Western印迹分析，在大鼠角叉菜胶性胸膜炎的胸液中检测到释放了缓激肽的HK分子，即无激动素的HK。结果表明，在炎症部位，激肽可能由HK释放，发挥炎症介质的作用。另一方面，尿激肽可以来源于血浆LK，因为激肽原缺陷的B/N-Katholiek大鼠在输注LK时尿激肽含量比HK显著增加。

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.