CHARACTERIZATION OF FUNDIC MUCOSAL CELLS IN VITRO

这项提案的总体目标是研究细胞机制。 胃酸胃酸的潜在分泌功能和粘膜抵抗 受伤。一个主要的焦点是使用细胞分离技术来识别 犬胃底粘膜中产生前列腺素(PG)的细胞。PGS将 用放射免疫法测定，用薄层层析法确认。 我们将验证我们最近的发现，分离的犬眼底巨噬细胞 (CFM)产生大量PG。监控因子VIII--相关 抗原，内皮细胞将被浓缩，PG的产生将被测定。 胃底巨噬细胞、血管内皮细胞和血管内皮细胞对PG形成的调节 粘膜细胞将被比较。我们将确认我们的初步调查结果 CFM产生超氧化物以响应吞噬刺激，并测试 CFM可能通过放氧介导胃损伤的假说 自由基、溶酶体酶、血栓素和白三烯。 将重点研究分泌细胞激活的机制。 环磷酸腺苷和钙离子的作用。胞浆内[Ca++]将通过 Quin2荧光技术和同位素通量研究。的作用 将研究肌醇磷脂在细胞激活过程中的周转 细胞内Ca~(++)的调节与蛋白质活化的关系 该方法将用于研究主细胞，然后 应用于顶叶细胞和生长抑素细胞，并酌情应用于眼底细胞 肥大细胞和眼底巨噬细胞。激活或激活之间的对比 各种化学递质对细胞功能的抑制将提供 用于确定这些细胞的特异性和相关性的关键工具 激活机制。 主要细胞单层的培养技术将得到改进，重点是 显影条件(底物、限定的无血清培养基和 条件培养基用来维持分化。这些单层 抵抗非常大的H+梯度，这一假设将得到检验 根尖表面的特化是屏障功能的基础。这个 主要细胞单层对H+反向扩散的抵抗力将通过 电生理和形态技术，以及与 非胃上皮细胞测定。主细胞基侧的作用 膜酸处理在细胞耐酸消化性损伤中的作用 探索过了。

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.