THE KININ RELEASED FROM HIGH-MOLECULAR WEIGHT-KININOGEN IS RESPONSIBLE FOR INFLAMMATORY EXUDATION IN RATS - DETECTION OF KININ-FREE-KININOGEN IN THE EXUDATE BY IMMUNOBLOT ANALYSIS

Kinin release and its involvement in inflammatory exudation were assessed by immuno-blot analysis of the kinin-precursor protein, high molecular weight kininogen (HK). HK consists of heavy (H) chain, bradykinin and light (L) chain. After bradykinin was released by plasma kallikrein, HK remains two-chain-kinin-free form;, i.e., H-chain and L-chain link each other through a disulfide bond. By Western blot analysis using antibody recognizing the light chain of HK, a band of 110-kD mass, which corresponds to intact HK, was detected in plasma after SDS-PAGE under reducing conditions, while a 46-kD band, corresponding to the light chain of HK, but no 110-kD band, was found in the exudate of rats with carrageenin-induced pleurisy at 3 hr as well as at 16 h. This result indicates that in the exudate most all of the HK molecules had released kinin to form kinin-free-HK, whereas the HK in the plasma remained intact. On the contrary, low molecular weight kininogen (LK) in the exudate was mostly in its intact form. These results indicate that plasma kallikrein could be activated in the exudate to release kinin from HK, as it reacts exclusively with HK and not with LK, and they are also mostly consistent with the features of the kinin release from the exudate and the plasma. That is, no kinin was detected in the exudate when the latter was incubated with plasma kallikrein, whereas salivary kallikrein did release kinin, indicating that kinin had already been released from HK, but not from LK in the exudate. Immunoblot analysis of HK in the pleural exudate also demonstrated no kinin involvement in phorbol myristate acetate- or zymosan-induced pleurisy, since no light chain band, but an intact HK band, was found in the exudates from these pleurisies.