AIM:To investigate the ability of agonists of PAR-2 to stimulate release of tryptase and histamine from human colon mast cells and the potential mechanisms.METHODS:Enzymatically dispersed cells from human colons were ...AIM:To investigate the ability of agonists of PAR-2 to stimulate release of tryptase and histamine from human colon mast cells and the potential mechanisms.METHODS:Enzymatically dispersed cells from human colons were challenged with tc-LIGRLO, tc-OLRGIL, SLIGKV,VKGILS, trypsin, anti-IgE or calcium ionophore A23187,and the cell supematants after challenge were collected. Tryptase release was determined with a sandwich ELISA procedure and histamine release was measured using a glass fibrebased fluorometric assay.RESULTS: Both PAR-2 agonists tc-LIGRLO-NH2 and SLIGKVNH2 were able to induce dose dependent release of tryptase and histamine from colon mast cells. More than 2.5 fold increase in both tryptase and histamine release was provoked by 100μmol/mL tc-LIGRLO-NH2, in comparison with only 2.0 fold increase being stimulated by SLIGKV-NH2，The reverse peptides tc-OLRGIL-NH2 and VKGILS-NH2 at the concentrations tested had no effect on the release of these two mediators.The maximum tryptase release elicited by tc-LIGRLO-NH2 was similar to that induced by anti-IgE(10μg/mL) or calcium ionophore (1μg/mL), though the latter was a more potent stimulus for histamine release.Both histamine and tryptase release in response to tc-LIGRLONH2 were completed within 3 rain. Trypsin at concentrations from 1.0 to 100μg/mL was capable of provoking a dose dependent release of tryptase as well as histamine with a maximum of 16ng/mL tryptase and 14ng/mL histamine release being achieved. An approximately 80% and 70% inhibition of trypsin induced release of tryptase and histamine were observed with SBTI, respectively. Pretreatment of cells with metabolic inhibitors or pertussis toxin abolished the actions of tc-LIGRLO-NH2, SLIGKV-NH2 and trypsin.CONCLUSION: The agonists of PAR-2 and trypsin are potent secretagogues of human colon mast cells, which are likely to contribute to the development of inflammatory disorders in human gut.展开更多
目的:研究PICK1(protein interacting with C kinase 1)蛋白PDZ结构域内83位点赖氨酸(K83)对PICK1和AMPA受体GluR2亚单位相互作用的影响。方法:利用计算机对PICK1 PDZ结构域和GluR2 C末端4个氨基酸残基进行对接模拟,然后将K83进行虚拟...目的:研究PICK1(protein interacting with C kinase 1)蛋白PDZ结构域内83位点赖氨酸(K83)对PICK1和AMPA受体GluR2亚单位相互作用的影响。方法:利用计算机对PICK1 PDZ结构域和GluR2 C末端4个氨基酸残基进行对接模拟,然后将K83进行虚拟点突变,计算并观察突变后结构和键能的改变。利用实验室已有的野生型全长PICK1 cDNA质粒为模板,构建点突变质粒,与野生型GluR2共转到HEK293T细胞,观察两者在细胞内定位和分布的改变。结果:当野生型PICK1与GluR2共转染时,HEK293T细胞有大量PICK1和GluR2共定位的集簇(cluster)。当我们把构建的PICK1突变体与GluR2共转染时,不同的突变体表现出不一样的改变。结论:改变K83位点的氨基酸结构,很可能会改变PICK1 PDZ结构域与GluR2 C末端结合所形成的疏水、氢键、静电相互作用,使得PDZ结构域与GluR2 C末端的结合能力发生不同程度的改变。展开更多
基金Supported by the National Natural Science Foundation of China,No.30140023,and the Li Ka Shing Foundation,Hong Kong,China,No.C0200001
文摘AIM:To investigate the ability of agonists of PAR-2 to stimulate release of tryptase and histamine from human colon mast cells and the potential mechanisms.METHODS:Enzymatically dispersed cells from human colons were challenged with tc-LIGRLO, tc-OLRGIL, SLIGKV,VKGILS, trypsin, anti-IgE or calcium ionophore A23187,and the cell supematants after challenge were collected. Tryptase release was determined with a sandwich ELISA procedure and histamine release was measured using a glass fibrebased fluorometric assay.RESULTS: Both PAR-2 agonists tc-LIGRLO-NH2 and SLIGKVNH2 were able to induce dose dependent release of tryptase and histamine from colon mast cells. More than 2.5 fold increase in both tryptase and histamine release was provoked by 100μmol/mL tc-LIGRLO-NH2, in comparison with only 2.0 fold increase being stimulated by SLIGKV-NH2，The reverse peptides tc-OLRGIL-NH2 and VKGILS-NH2 at the concentrations tested had no effect on the release of these two mediators.The maximum tryptase release elicited by tc-LIGRLO-NH2 was similar to that induced by anti-IgE(10μg/mL) or calcium ionophore (1μg/mL), though the latter was a more potent stimulus for histamine release.Both histamine and tryptase release in response to tc-LIGRLONH2 were completed within 3 rain. Trypsin at concentrations from 1.0 to 100μg/mL was capable of provoking a dose dependent release of tryptase as well as histamine with a maximum of 16ng/mL tryptase and 14ng/mL histamine release being achieved. An approximately 80% and 70% inhibition of trypsin induced release of tryptase and histamine were observed with SBTI, respectively. Pretreatment of cells with metabolic inhibitors or pertussis toxin abolished the actions of tc-LIGRLO-NH2, SLIGKV-NH2 and trypsin.CONCLUSION: The agonists of PAR-2 and trypsin are potent secretagogues of human colon mast cells, which are likely to contribute to the development of inflammatory disorders in human gut.
