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THE INTESTINAL-CELL MONOLAYER OF THE DOG (CANIS FAMILIARIS) AND MALPIGHIAN TUBULES OF AEDES AEGYPTI: POWERFUL IN VITRO MODELS FOR STUDYING EPITHELIAL TRANSPORT
Homeostasis of the internal environment of vertebrates and invertebrates is maintained by a collection of organs involving the gastrointestinal system, the renal system and the pulmonary system, whose surfaces facing the external world are covered by a layer of highly polarized epithelial cells as the barrier and sites for substance exchange between body and the external world. As most studies on epithelial transport are done in animal cell- or tissue-models, this dissertation is set to address questions on two in vitro epithelial models: monolayers of cultured epithelial cells and Malpighian tubules of insects. In the field of epithelial transport across mammalian small intestines, most of the cell monolayers that have been used in the past are abnormal: they are either derived from cancerous cells or poorly differentiated compared to normal tissues. By strictly controlling cultural conditions, we successfully developed an epithelial cell culture derived from normal dog jejune with full differentiation, which can form cell monolayers with remarkable resemblance to the normal small intestinal epithelia in morphology, expression of the junctional proteins and membrane transporter, and electrophysiological properties. Furthermore, different channels can be induced when the cell monolayer is treated with different hormones and agents, suggesting its wide application in basic and applied research studies. Malpighian tubule of the yellow fever mosquitoes Aedes aegypti has been the epithelial transport model in our lab for more than twenty years. The V-type H+ ATPase is thought to provide driving force for transepithelial electrolyte and fluid secretion in Malpighian tubules. Here we immunohisochemcially localized the proton pumps to the apical membranes of the mitochondria-rich principal cells of the Malpighian tubules, and measured enzymatic activities of the V-type H+ ATPase as 50~60% of the total ATPase activities. In comparison the activities of the Na+/K+ ATPase were almost undetectable. To understand the functions of Malpighian tubules, we also modeled the tubule into equivalent electrical circuits. However in our previous studies, the model was incomplete, as contribution of gap junction resistance between Malpighian tubule cells to the circuit has never been revealed. In this dissertation, we accurately measured the electrical resistance of gap junctions in a single cell by circuit analysis and rectified the values of all the electrical elements in our previous model. The equivalent electrical circuit has for the first time become complete. The results also show that gap junctions in Aedes Malpighian tubules are impermeable to the fluorescent dye Lucifer yellow CH, sensitive to the metabolic inhibitor dinitrophenol, while not so sensitive to elevated intracellular [Ca2+].