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 i s 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 i s 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 i s 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.
