SALT AND WATER TRANSPORT IN PANCREAS DUCT EPITHELIUM

外分泌胰腺分泌消化酶和等渗液。 消化酶被包装并储存在其中，然后从 酶原颗粒位于腺泡细胞内。在大多数物种中，腺泡 细胞还会分泌少量等渗的氯化钠溶液到 流明。相比之下，导管上皮细胞只包含 大约5%的胰腺肿块会分泌大量的 等渗溶液。胰液是碱性的，[HCO3-]范围 从老鼠的大约60毫米到猪的大约150毫米。小才是 已知盐和水通过胰腺的转运机制 导管上皮。这项研究提案的目标是开始 在细胞水平上理解盐和水的作用机制 导管上皮细胞的分泌物。跨上皮电和离子 通量实验，结合流体传输测量，将 以确定其基线和受刺激的传输特性 导管细胞。细胞内微电极实验将进行 在单膜水平上确定电驱动力和 确定电扩散渗透率。细胞内离子活度 将使用离子敏感微电极和/或通过使用 荧光细胞内离子指示剂。这样的测量将提供 在单一的膜水平上，关于大小和 化学驱动力的方向。此外，中国的变化 细胞内离子活性对实验扰动的响应将 用于识别电中性传输机制(例如， 交易所、联运公司)。膜片钳技术将用于 确定并详细研究受以下因素影响的渠道 细胞内微电极研究作为生物医学研究的重要组成部分 跨上皮离子转运。一种干管原代细胞培养方法 将使用上皮细胞，因为：1)来源于 胰腺肿瘤分化低，总体上不确定。 起源(导管与腺泡)，2)胰腺的复杂形态使 用小导管进行实验在技术上是困难的，细胞培养将 允许生物材料的放大用于生化和 分子生物学研究；4)途径和技术 为研究牛胰腺而开发的应直接适用 对人类胰腺分泌盐分和水分的研究。这项研究 将有助于我们对离子传输的理解 通过哺乳动物胰腺导管上皮细胞，并提供了一种模型系统 HCO3分泌调节的研究。开发的方法和 从这些实验中获得的信息将直接 适用于人正常胰腺和病变胰腺的类似研究。 这与囊性纤维化尤其相关，在这种疾病中 胰腺的盐分和水分分泌严重受损。

The exocrine pancreas secretes digestive enzymes and an isosmotic fluid. The digestive enzymes are packaged and stored in, and released from zymogen granules located in acinar cells. In most species the acinar cells also secrete a small volume of isosmotic NaCl solution into the lumen. In contrast, the ductal epithelial cells which comprise only approximately 5% of the mass of the pancreas secrete a large volume of an isosmotic solution. Pancreatic juice is alkaline and the [HCO3-] ranges from approximately 60 mM in rat to approximately 150 mM in pig. Little is known about the mechanisms of salt and water transport across pancreatic ductal epithelium. It is the goal of this research proposal to begin to understand at the cellular level the mechanisms of salt and water secretion by ductal epithelial cells. Transepithelial electrical and ion flux experiments, in conjunction with fluid transport measurements, will be done to determine baseline and stimulated transport properties of ductal cells. Intracellular microelectrode experiments will be done to determine at the single membrane level electrical driving forces and to identify electrodiffusive permeabilities. Intracellular ion activities will be measured with ion sensitive microelectrodes and/or by the use of fluorescent intracellular ion indicators. Such measurements will provide information, at the single membrane level, about the magnitude and direction of chemical driving forces. Furthermore, changes in intracellular ion activity in response to experimental perturbations will be used to identify electrically neutral transport mechanisms (e.g., exchangers, cotransporters). Patch clamp techniques will be used to identify and study in detail channels that are implicated by intracellular microelectrode studies as important components of transepithelial ion transport. A primary cell culture of main duct epithelial cells will be used because: 1) cell lines derived from pancreatic tumors are poorly differentiated and in general of uncertain origin (ductal vs. acinar), 2) the complex morphology of pancreas makes experiments with small ducts technically difficult, 3) cell culture will allow for amplification of biologic material for biochemical and molecular biological studies, and 4) the approaches and techniques developed for the study of bovine pancreas should be directly applicable to the study of salt and water secretion by human pancreas. The research outlined in this proposal will aid in our understanding of ion transport by mammalian pancreatic ductal epithelium and provide a model system for the study of regulation of HCO-3 secretion. The methods developed and the information obtained from these experiments will bc directly applicable to similar studies with normal and diseased human pancreas. This is particularly relevant to cystic fibrosis, a disease in which pancreatic salt and water secretion are severely compromised.