The work in this proposal will advance the understanding of the kallikrein-kinin system and its role(s) in the regulation of epithelial ion transport, a process important to the maintenance of normal cell function. Cystic fibrosis is now thought to result from an epithelial defect in chloride transport, e.g., and secretory diarrhea from inappropriate regulation of the same process. Ion-transporting phenomena also contribute to aspects of blood pressure control and diabetes, and aberrancies in the kallikrein-kinin system have been associated with both of these diseases.
The specific aims of this proposal will establish whether the kallikrein-kinin system participates as an autocrine or paracrine regulator of chloride transport in T84 cells, a human colonic epithelial cell line derived from an adenocarcinoma. The system in these cells will be activated or inhibited, and the effects on chloride movements monitored in the Ussing chamber. This will be accomplished (1) by activating or inhibiting the protease kallikrein with appropriate agents, and establishing the specificity of the results by blocking the actions of the released kinin peptide at its receptors, and (2) by increasing the activity of the system using physiological signals such as growing the cells as a tumor in the nude mouse, or increasing the extracellular potassium concentration, treatments that increase kallikrein production and kinin sensitivity. The molecular mechanisms underlying the physiologically-induced changes in the kallikrein-kinin system will also be investigated. 125I-bradykinin binding studies will measure changes in kinin receptor number and/or affinity, and determinations of calcium mobilization and inositol phosphate synthesis will measure changes in second messenger production. Kallikrein (or kallikrein like) gene expression will be studied by evaluating mRNA transcripts, and the role of the Na+-K+-Cl- cotransport system in the IV effect will be examined by inhibiting the cotransport with bumetanide prior to a potassium challenge. The regulation of ion transport in epithelia is complex, involving multiple neural and hormonal influences. Elucidation of the contribution of the kallikrein-kinin system to these processes will reduce this complexity. These findings will be relevant not only to the definition of fundamental cellular processes, but also to the understanding of human disease.
