The aims of this project are to identify the Cl- channels in the apical and basolateral membranes of the epithelial cells of the gallbladder of Necturus maculosus and to determine the mechanisms underlying their physiological regulation and pathophysiological alterations. Our focus is on the ion-transport function of the epithelial cell, which involves the coordinated action of transporters expressed in the cell membranes. Our strategy is to compare data obtained from the entire epithelium, single cells and membrane patches. Comparison of the experimental results obtained at these levels allows for unique cell-physiological interpretation of biophysical results. The experimental methods include intracellular microelectrodes, patch clamp, quantitative fluorescence microscopy and molecular biology. The choice of regulation of apical- membrane Cl- channels as the main problem is based on the following arguments: a) a 10-pS channel expressed in this membrane is functionally similar to the cystic fibrosis transmembrane conductance regulator (CFTR) and may be a CFTR homologue, b) there is a possibility of coexpression of other Cl- channels postulated to be regulated by CFTR, c) the NGB epithelium could be a good prototype for absorptive epithelia expressing a CFTR-like channel, and d) the effects of cAMP on epithelial transport are not completely understood. Experiments will be designed to ascertain the mechanisms of the effects, on Cl- channels, of protein kinase C, neurotransmitters, such as vasoactive intestinal peptide (VIP) and norepinephrine, and mediators of inflammation, such as bradykinin and prostaglandins. Other studies will examine the regulation of basolateral- membrane Cl- channels, in particular the effect of HCO3-/CO2, the mechanisms of cross-talk between apical and basolateral membranes, and the roles of cell volume, intracellular ionic activities and membrane voltage. These studies are expected to provide important new information on the mechanisms of regulation of ion transport in gallbladder, in other biliary-tract epithelia and in absorptive epithelia such as those of the intestine and the kidney.
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