INTESTINAL SECRETORY MECHANISMS

此前，我们的实验室已经鉴定出一种人结肠上皮细胞。 品系T84作为研究氯离子分泌的合适模型 机械装置。细胞系，只由分泌细胞组成，不含 旁分泌、神经分泌或内分泌控制的影响是理想的 细胞水平上氯离子分泌调控的研究。我们的 在过去的四年中，研究确定了涉及 不同的氯离子分泌过程和揭示的协同现象 环核苷酸和钙相关分泌物之间的关系。最新研究 提示Ca++-中存在更复杂的二级信使相互作用 磷脂的相关分泌机制及其隐含参与 失误。一种候选信使(磷脂代谢物)，它与 用胞内游离钙离子花生四烯酸激活氯离子分泌。一个 抑制氯离子分泌的候选信使是甘油二酯。这 续展申请建议梳理二手房的贡献 信使，特别是利用细胞单层和磷脂代谢物 质膜制剂。将使用三种方法：1) 每种药物的时间进程和剂量反应的相关性研究 辅助信使和识别潜力的生物反应 候选人。磷脂酶激活剂或抑制剂--二甘油酯 脂肪酶、脂氧合酶、环氧合酶或G蛋白也将用于 不同[Ca++]i或[Ca++](质膜)条件下的研究。首字母 研究将集中在卡巴胆碱、组胺、4-氨基丁酸的作用机制上。 BR-A23187、离子霉素、牛磺酸脱氧胆酸和腺苷。2)重组 研究将在一种更好的输送脂溶物质的方法之后进行 物质向细胞或质膜发展。生物学的 每个候选信使的动作将分别单独和 与其他人结合起来模仿每个促分泌剂的反应 已列入相关研究。同样，使用特定的酶和 它们的抑制剂将补充在不同[Ca++]i下提出的研究 或[Ca++]。3)将在其他上皮性组织中进行比较研究 分泌氯离子的细胞系，例如Caco和HT-29克隆，以测试 同样的机制也适用于它们。预计会有一些细胞 次级信使缺陷或运输途径缺陷的品系 将为信使的角色提供有用的补充见解 在运输路线上。

Previously, our laboratory has identified a human colonic epithelial cell line, T84, as a suitable model for investigation of Cl- secretory mechanisms. The cell line, composed of only secretory cells and free of influence from paracrine, neurocrine or endocrine controls, is ideal for studies of regulatory controls of Cl- secretion at the cellular level. Our studies in the past four years identified transport pathways involved in various Cl- secretory processes and unmasked a synergistic phenomenon between cyclic nucleotide and Ca++ -related secretions. Recent studies suggest a more complicated interaction of secondary messengers in Ca++ - related secretory mechanisms and implicate involvement of phospholipid turnovers. A candidate messenger (phospholipid metabolite) which co- activates Cl- secretion with free cytosolic Ca++ is arachidonic acid. A candidate messenger which inhibits Cl- secretion is diacylglycerol. This renewal application proposes to sort out the contribution of secondary messengers, particularly phospholipid metabolites using cell monolayers and plasma membrane preparations. Three approaches will be utilized: 1) Correlative studies of the time courses and dose-responses on each secondary messenger and a biological response to identify potential candidates. Activators or inhibitors of phospholipases, diglyceride lipase, lipoxygenase, cyclooxygenase or G-proteins will also be used in the studies under varying [Ca++]i or [Ca++] (for plasma membrane). Initial studies will focus on the mechanism of action of carbachol, histamine, 4- Br-A23187, ionomycin, taurodeoxy-cholate and adenosine. 2) Reconstitution studies will be carried out after a better method to deliver fat-soluble substances to the cell or plasma membrane is developed. The biological actions of each candidate messenger will be directly tested both alone and in combination with others to mimic the responses of each secretagogue listed in correlative studies. Again, the use of specific enzymes and their inhibitors will complement the studies proposed under varying [Ca++]i or [Ca++]. 3) Comparative studies will be carried out in other epithelial cell lines that secrete Cl-, e.g., CaCO and HT-29 clones to test whether the same mechanism also applies to them. It is expected that some cell lines with a defective secondary messenger or a defective transport pathway will provide useful complementary insights into the role of that messenger on the transport pathway.