Effects of chymase on limited proteolysis and activation of protein kinase on the mechanism of degranulation from mast cells

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Two types of serine proteases, such as chymase and tryptase, are localized in the grnules of mast cells. We found that antibody of chymase, substrate analogues and specific inhibitor of chymase inhibited histamine release from rat mast cells but that inhibitors of tryptase did not. In addition specific inhibitors of chymase and anti-chymase F(ab')_2 also inhibited protein phosphorylation by protein kinase C after bridging of IgE-receptor. From these results, We speculate that chumase activates protein kinse C on the process of degranulation. After degranulation, chymase retained on the surface of mast cells. In order to clarify which of the chymase in the granules or on the cell surface is involved in histamine release, we examined the intra-cellular localization of the inhibitores of both histamine release and chymase, such as antichymase f(ab')_2 and ^<125>I-Bowman Burk soybean inhibitor. After incubation of these inhibitors with mast cells at 37゜C, they first bound to plasma membrane of mast cells and then were incorporated into the granules time-dependently, resulting in the inhibition of histamine release after bridging of IgE receptor. When these inhibitors were incubated with mast cells at 4゜C, they bound to plasma membrane but did not incorporated into the granules and did not also inhibit histamine release. These results show that incorporation of these inhibitors into the granules, in which chymase is localized, is essential for inhibition of histamine release by these inhibitors.Studies on the mechanisms of activation of protein kinase C by chymase and of protein phosphorylation by the activated protein kinase C are now in progress.