CL TRANSPORT REGULATION IN CULTURED HUMAN COLONOCYTES

这项建议将表征中国对C1运输的独特调控 人结肠正常细胞原代培养及其差异研究 沿着头尾轴线。从动物物种到人类的各种模型 结肠癌细胞系已被用来了解人结肠C1 运输。动物模型并不总是适用于人类，因为 结肠离子沿头尾的运输是否存在物种差异？ 轴心。人结肠癌细胞株不能用于节段性研究 不同之处。此外，作为“转化”的细胞，它们经常 显示小肠功能优于结肠功能。认识 这些限制，细胞内C1转运调节的复杂性 从这些模型中辨别出来的将必须与主要事件相关 人类结肠细胞的培养，并最终移植到那些完好无损的， 正常，冒号。这个实验室已经开发出24小时的原代培养 人结肠细胞对cAMP、Ca~(2+)和cGMP敏感的C1转运， 与其他结肠相比，更接近完整结肠的特征 模特们。与结肠癌细胞株相比，T-84、C1在人体内的转运 克隆细胞可以被佛波酯和人激活，但不能被兔子激活， 结肠细胞有cGMP激活的C1转运。因此，假设 有待检验的是细胞对C1转运的调节作用 人类结肠细胞的培养与完整的人类结肠细胞相似， 显示分段差异，并与其他模型中看到的不同 系统。第一个目标是定义c1中的独特差异 不同结肠段细胞的通透性。节约能源 组织，荧光技术将被用于研究离子的特性， 对抑制剂和动力学的敏感性。细胞外环境对环境的影响 将研究关于C1输运的矩阵(ECM_)。第二个目标是 确定cAMP、Ca~(2+)和cGMP对CI通透性的调节是否 受节段差异和/或ECM的影响。的具体方面 CGMP和蛋白激酶C(PKC)级联将被分析为它们的 在不同的结肠细胞模型中，存在/作用不同。会是 已确定cGMP是通过cAMP-PK还是cGMP-PK起作用，以及是否存在 阵营之间的串扰？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及其在细胞内的变化 转运蛋白Cftr和Na-K-2c1的磷酸化 辅助传送器。第三个目标是将C1的传输特性联系起来 人结肠上皮细胞与结肠上皮细胞的培养 结肠上皮片，在手术时获得。具体来说， 第二信使激活的节段差异和串扰 将研究调节跨上皮细胞C1运输的系统。这些 研究将提供有关正常结肠细胞的宝贵信息 生理学，可以用来理解分子病理生理学。 毒素引起的腹泻、囊性纤维化和炎症性疾病。

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.