AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ducta...AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 μmol/L carbachol evoked a dose- dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.展开更多
目的研究突触小体相关蛋白(snaptosome associated protein of 23 KD,Snap23)与Munc 18C蛋白的相互作用。方法从人肌肉组织中钓取人Snap23基因并克隆入pEYFP-N1载体中,构建pEYFP-Snap23质粒,瞬时转染入CHO细胞后荧光显微镜观察并拍照,...目的研究突触小体相关蛋白(snaptosome associated protein of 23 KD,Snap23)与Munc 18C蛋白的相互作用。方法从人肌肉组织中钓取人Snap23基因并克隆入pEYFP-N1载体中,构建pEYFP-Snap23质粒,瞬时转染入CHO细胞后荧光显微镜观察并拍照,免疫蛋白印迹检测EYFP-Snap23蛋白的表达。共转染pEYFP-Snap23和PECFP-Munc 18c质粒,利用荧光共振能量转移的方法检测二者是否有相互作用,同时共转染pEYFP-Snap23和pECFP-N1作为对照组。结果成功构建pEYFP-Snap23载体,荧光共振能量转移实验证明Snap23与Munc 18C蛋白有相互作用。结论突触小体相关蛋白Snap23可能与Munc 18C相互作用。展开更多
基金Grants to H.Y.G. from the U.S. National Institute of Health, R21 AA015579-01A1 and to S.V. form the Canadian Institute of Health Research
文摘AIM: To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 μmol/L carbachol evoked a dose- dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.
文摘目的研究突触小体相关蛋白(snaptosome associated protein of 23 KD,Snap23)与Munc 18C蛋白的相互作用。方法从人肌肉组织中钓取人Snap23基因并克隆入pEYFP-N1载体中,构建pEYFP-Snap23质粒,瞬时转染入CHO细胞后荧光显微镜观察并拍照,免疫蛋白印迹检测EYFP-Snap23蛋白的表达。共转染pEYFP-Snap23和PECFP-Munc 18c质粒,利用荧光共振能量转移的方法检测二者是否有相互作用,同时共转染pEYFP-Snap23和pECFP-N1作为对照组。结果成功构建pEYFP-Snap23载体,荧光共振能量转移实验证明Snap23与Munc 18C蛋白有相互作用。结论突触小体相关蛋白Snap23可能与Munc 18C相互作用。
