Alic, Arna; (2001) Involvement of proteases and kinases in mast cell activation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Activation of non-receptor protein tyrosine kinases (PTK) and a chymotryptic serine esterase (SE) zymogen are among the earliest events in the FcεRI signalling process. The aim of this study was to clarify the individual roles of these enzymes in immunological signal transduction of human basophils and mast cells from different species. Using a range of inhibitors, we confirmed that PTK activation is a step common to the immunological signal transduction of all histaminocytes. Of the test agents, ST638 was most selective in blocking IgE-mediated histamine release and was used, together with the protein phosphatase (PP) inhibitor okadaic acid, to examine the possibility that PTK is implicated in activating the SE zymogen. Immunological activation of mast cells loaded with a fluorescent chymotryptic substrate led to a sharp rise in fluorescence, indicating activation of the zymogen and hydrolysis of substrate. The fluorescence changes were markedly reduced by pretreatment with inhibitors of chymotrypsin and PTK, indicating an interrelationship between the kinase and the esterase in the signalling process. Ligands for the known protease activated receptors (PAR) failed to induce histamine release from mast cells and basophils. However, a range of proteases with differing substrate specificities, including α-chymotrypsin, were found to evoke a non-cytotoxic release of histamine from the former cells. The augmented histamine release was coupled to an increase in cytosolic calcium. It is therefore possible that mast cells possess a novel PAR which is activated by chymotryptic and other proteases. The histamine release induced by the proteases was reduced by inhibitors of SE, PTK and PP, suggesting that they trigger a signalling cascade which involves non-receptor PTK and chymotryptic SE. Genistein and selected proteases were also found to cause relaxation of pre-contracted ileal smooth muscle, suggesting a role for these agents in stimulus-contraction as well as stimulus-secretion coupling.

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