CL TRANSPORT REGULATION IN CULTURED HUMAN COLONOCYTES

该提案将描述C1运输的独特调节， 原代培养的正常人结肠细胞，并研究其差异 沿着头尾轴。 从动物物种到人类的模型 结肠癌细胞系已被用于了解人类结肠C1 运输 动物模型并不总是适用于人类，因为 是结肠离子转运沿着头尾向的种属差异 轴线 人结肠癌细胞系不能用于研究节段性 差异 此外，作为“转化”细胞，它们通常 小肠功能优于结肠功能。 认识 这些限制，细胞C1运输调节的复杂性 从这些模型中辨别出来的信息必须与主要事件有关， 人结肠细胞的培养物，并最终到那些完整的， 正常结肠 该实验室已开发出原代24小时培养物， 显示cAMP、Ca 2+和cGMP敏感性C1转运的人结肠细胞， 与完整结肠相比， 模型 与结肠癌细胞系T-84相比， 结肠细胞被佛波醇酯和人激活，但不是兔， 结肠细胞具有cGMP激活的C1转运。 因此，假设 要测试的是，细胞调节C1运输在初级 人结肠细胞的培养物类似于完整的人结肠， 显示了节段差异，与其他模型中所见不同 系统. 第一个目标是定义C1中的独特差异 在来自不同结肠节段的细胞中的渗透性。 以节省 组织，荧光技术将用于研究离子特异性， 对抑制剂和动力学的敏感性。 细胞外的影响 基质（ECM_）对C1转运的影响。 第二个目标是 确定cAMP、Ca 2+和cGMP对Ci渗透性的调节是否是 受节段差异和/或ECM的影响。 的具体方面 cGMP和蛋白激酶C（PKC）级联将被分析， 存在/作用在不同的结肠细胞模型中不同。 将 确定cGMP是否通过cAMP-PK或cGMP-PK起作用，以及是否存在 cross-talk之间的cAMP？PKC级联。 这些步骤中的顺序步骤 将检查级联反应，包括细胞内介质的变化， 蛋白激酶和磷酸酶抑制剂的作用以及 CFTR和Na+-K+-2C1等转运蛋白的磷酸化 协同转运蛋白 第三个目标是将C1运输特性 使用人结肠细胞与完整上皮细胞培养， 结肠上皮片，在手术时获得。 具体地说， 第二信使激活的 将研究调节跨上皮C1转运的系统。 这些 研究将提供关于正常结肠细胞的宝贵信息， 生理学，可用于了解分子病理生理学， 毒素介导的子宫内膜异位症、囊性纤维化和炎性疾病。

This proposal will characterize the unique regulation of C1 transport in normal, human colonocytes in primary cultures and study its differences along the cephalocaudal axis. Models ranging from animal species to human colon carcinoma cell lines have been used to understand human colonic C1 transport. Animal models are not always applicable to humans since there are species differences in colonic ion transport along the cephalocaudal axis. Human colon carcinoma cell lines cannot be used to study segmental differences. In addition, being "transformed" cells, they often demonstrate small intestinal better than colonic function. Recognizing these limitations, the complexities of cellular C1 transport regulation discerned from these models will have to be relate to events in primary cultures of human colonocytes and ultimately to those in the intact, normal, colon. This laboratory has developed primary, 24 hr cultures of human colonocytes which exhibit cAMP, Ca2+ and cGMP-sensitive C1 transport, features that resemble the intact colon more closely than other colonic models. In contrast tot he colonic cell line, T-84, C1 transport in human colonocytes is activated by phorbol esters and human, but not rabbit, colonocytes have a cGMP-activated C1 transport. Therefore, the hypothesis to be tested is that the cellular regulation of C1 transport in primary cultures of human colonocytes resembles that of the intact human colon, shows segmental differences and is distinct from that seen in other model systems. The first aim is to define the unique differences in C1 permeabilities in cells from different colonic segments. To conserve tissues, fluorimetric techniques will be used to study ion specificities, sensitivity to inhibitors and kinetics. The influence of extracellular matrices (ECM_) on C1 transport will be studied. The second aim is to determine if the regulation of Ci permeabilities by cAMP, Ca2+ and cGMP, is influenced by segmental differences and/or by the ECM. Specific aspects of the cGMP and protein kinase C (PKC) cascades will be analyzed as their presence/role varies amongst the different colonocyte models. It will be determined if cGMP is acting via a cAMP-PK or a cGMP-PK and if there is cross-talk between the cAMP?PKC cascades. Sequential steps in these cascades will be examined including changes in intracellular mediators, the effects of inhibitors of protein kinases and phosphatases and changes in phosphorylation of transporters such as CFTR and the Na+-K+-2C1 cotransporter. The third aim is to relate the C1 transport characteristics of colonocytes in culture with those of the intact epithelium using human colonic epithelial sheets, obtained at the time of surgery. Specifically, segmental differences and cross-talk between second messenger-activated systems in regulating transepithelial C1 transport will be studied. These studies will provide invaluable information about normal colonic cell physiology which can be used to understand the molecular pathophysiology of toxin-mediated diarrheas, cystic fibrosis an inflammatory disorders.