GF REGULATION/GASTRIC MUCOSAL PARACELLULAR PERMEABILITY

说明：本提案的长期目标是确定 上皮机制，使胃粘膜细胞生存在 酸性消化环境。 犬胃细胞原代培养模型， 其中单层显示对顶端酸化的抗性。 先前的工作确定细胞旁途径是主要位点 对H离子的抵抗力，以及过度暴露的早期损伤部位 酸。 初步数据显示，内源性生长因子减少 细胞旁渗透性和增强对顶端酸的耐受性。 EGF的作用 顶侧和基底侧受体，而胰岛素样生长因子-I （IGF-I）和碱性成纤维细胞生长因子（bFGF）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快速的 通过激活选择性的 信号转导途径 生长因子对细胞旁 渗透性和屏障功能，通过跨上皮阻力测量 甘露醇和菊糖的通量进行比较。 添加 针对任一侧的免疫中和或受体阻断抗体将 确认这些影响的特异性，并确定内源性因素 来调节渗透性。 生长因子依赖性酪氨酸调节 磷酸化、顶端/连接分布和蛋白质相互作用 将使用以下方法检查连接组件之间的连接 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调节的作用。 最后，所选信号分子的激活和相关性可能 介导生长因子调节细胞旁通透性和酪氨酸 磷酸化（例如，磷脂酰肌醇-3激酶或Rho GTP结合 蛋白质）将通过使用特异性抑制剂和活化剂来评估。 这些研究应该允许结构、功能和分子 顶端连接调节的基本过程的表征 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.