GF REGULATION/GASTRIC MUCOSAL PARACELLULAR PERMEABILITY

描述：这项提案的长期目标是确定 使胃粘膜细胞在胃粘膜中存活的上皮机制 酸性的消化性环境。建立了犬胃细胞原代培养模型。 开发的单层对顶端酸化表现出抵抗力。 以前的工作证实，细胞旁途径是主要的部位 对H离子的抵抗力，以及过度暴露的早期损伤部位 变酸了。初步数据显示，内生增长因素减少 细胞旁通透性和增强对心尖酸的耐受性。EGF作用于 心尖和基底外侧受体，而胰岛素样生长因子-I 胰岛素样生长因子-I和碱性成纤维细胞生长因子仅通过 基底外侧受体。拟议中的实验将检验这一假设。 生长因子对细胞旁通透性的调节涉及到快速 选择性激活对心尖连接复合体的调节作用 信号转导通路。生长因子对细胞旁的影响 通透性和屏障功能，由跨上皮阻力测量 并通过甘露醇和菊粉的流量进行比较。添加 针对任何一方的免疫中和抗体或受体阻断抗体将 确认这些影响的特殊性并确定内生因素 调节渗透性的物质。生长因子对酪氨酸的调节作用 磷酸化、心尖/连接分布和蛋白质相互作用 将使用以下工具检查连接组件之间的 免疫沉淀/蛋白质印迹分析，共聚焦显微镜和 免疫共沉淀。此外，肌动蛋白聚合抑制剂将 用于评估生长因子对细胞骨架的调节作用。 最后，选定的信号分子的激活和相关性可能 生长因子对细胞旁通透性和酪氨酸的调节作用 磷酸化(例如，磷脂酰肌醇-3激酶或Rho GTP结合 蛋白质)将通过使用特定的抑制物和激活剂进行评估。 研究应该允许结构、功能和分子方面的研究 根尖连接调节基本过程的表征 在与胃上皮生理学相关的原代细胞模型中。

DESCRIPTION: The long-term objectives of this proposal are to identify epithelial mechanisms that enable gastric mucosal cells to survive in the acid peptic environment. A primary canine gastric cell culture model was developed in which monolayers display resistance to apical acidification. Previous work established that the paracellular pathway is the primary site of resistance to H ions, and an early site of injury from excessive exposure to acid. Preliminary data show that endogenous growth factors decrease paracellular permeability and enhance tolerance to apical acid. EGF acts on apical and basolateral receptors whereas insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) act only through basolateral receptors. The proposed experiments will test the hypothesis that growth factor regulation of paracellular permeability involves rapid modulation of the apical junctional complex via activation of selective signal transduction pathways. Growth factor effects on paracellular permeability and barrier function, as measured by transepithelial resistance and by mannitol and inulin flux, will be compared. Addition of immunoneutralizing or receptor-blocking antibodies to either side will confirm the specificity of these effects and identify endogenous factors that regulate permeability. Growth factor-dependent modulation of tyrosine phosphorylation, apical/junctional distribution, and protein interactions among junctional components will be examined using immunoprecipitation/western blot analysis, confocal microscopy and co-immunoprecipitation. In addition, actin polymerization inhibitors will be used to assess the role of cytoskeletal regulation by growth factors. Lastly, activation and relevance of selected signaling molecules potentially mediating growth factor regulation of paracellular permeability and tyrosine phosphorylation (e.g., phosphatidylinositol-3 kinase or the Rho GTP binding proteins) will be assessed by use of specific inhibitors and activators. The studies should allow structural, functional and molecular characterization of the fundamental process of apical junctional regulation in a primary cell model with relevance to gastric epithelial physiology.