CHARACTERIZATION OF FUNDIC MUCOSAL CELLS IN VITRO

这项提案的总体目标是研究细胞机制 潜在的分泌功能和粘膜对酸消化的抵抗力 损伤 一个主要的焦点是使用细胞分离技术来鉴定 在犬胃底粘膜中产生胰高血糖素（PG）的细胞。 PG将 通过放射免疫测定法测定，并通过薄层色谱法确认。 我们将证实我们最近的发现，分离的犬胃底巨噬细胞 (CFM)产生大量的PG。 监测因子VIII相关 抗原后，将富集内皮细胞并测定PG产生。 胃底巨噬细胞、内皮细胞和巨噬细胞对PG形成的调节 将比较粘膜细胞。 我们会确认我们的初步发现 CFM对吞噬刺激产生超氧化物，并测试 CFM可能通过释放氧介导胃损伤的假说 自由基、溶酶体酶、血栓素和白三烯。 分泌细胞激活的机制将被研究，重点是 cAMP和Ca++的作用。 将使用以下方法监测细胞质[Ca++] Quin 2荧光技术和同位素通量研究。 的作用 细胞活化中的磷酸肌醇周转将在 与调节胞浆Ca++和蛋白质活化的关系 激酶C 该方法将通过研究主细胞来开发， 应用于壁细胞和生长抑素细胞，并酌情应用于胃底细胞。 肥大细胞和胃底巨噬细胞。 激活或 通过各种化学递质抑制细胞功能将提供 确定这些细胞特异性和相关性的关键工具 激活机制 主细胞单层的培养技术将得到改进，重点是 显色条件（底物、确定成分的无血清培养基和 条件培养基）以维持分化。 这些单分子层 抵抗非常大的H+梯度，并将测试假设， 顶端表面的特化是屏障功能的基础。 的 主细胞单层对H+反向扩散的阻力将通过以下方法进行研究： 电生理学和形态学技术， 非胃上皮细胞测定。 基底外侧主细胞的作用 细胞膜酸处理对胃酸的抵抗-消化道损伤将是 探讨了

The overall objectives of this proposal are to study cellular mechanisms underlying secretory function and mucosal resistance to acid-peptic injury. A major focus is to use cell separation techniques to identify the cells producing prostaglandins (PG) in the canine fundic mucosa. PGs will be measured by radioimmunoassay, confirmed by thin layer chromatography. We will verify our recent finding that isolated canine fundic macrophages (CFM) produce large amounts of PGs. Monitoring Factor VIII-related antigen, endothelial cells will be enriched and PG production determined. The regulation of PG formation by fundic macrophages, endothelial cells and mucosal cells will be compared. We will confirm our preliminary finding that CFM produce superoxides in response to phagocytic stimuli, and test the hypothesis that CFM may mediate gastric injury via release of oxygen radicals, lysosomal enzymes, thromboxane and leukotrienes. The mechanisms of secretory cell activation will be studied focusing on the role of cyclic AMP and Ca++. Cytoplasmic [Ca++] will be monitored using Quin2 fluorescence techniques and isotopic flux studies. The role of phosphoinositide turnover in cell activation will be studied in relationship to modulation of cytosolic Ca++ and activation of protein kinase C. The approach will be developed studying chief cells and then applied to parietal and somatostatin cells and, as appropriate, to fundic mast cells and fundic macrophages. Contrasts between activation or inhibition of cell function by various chemotransmitters will provide the critical tool for establishing the specificity and relevance of these cell activation mechanisms. Culture techniques for chief cell monolayers will be refined, focusing on developing conditions (substrates, defined serum-free medium, and conditioned medium) for maintaining differentiation. These monolayers resist a very large H+ gradient and the hypothesis will be tested that specialization of the apical surface underlies barrier function. The resistance of chief cell monolayers to H+ backdiffusion will be studied by electrophysiological and morphological techniques, and differences with nongastric epithelial cells determined. A role for chief cell basolateral membrane acid disposal in cellular resistance to acid-peptic injury will be explored.