We intend to study the regulation of ENaC and the basolateral Na/K- ATPase under conditions of chronic up regulation or down regulation of apical Na+ entry. Although many studies have looked at short term regulation, few studies address chronic regulation of these transporters. Furthermore, the advent of an in vitro model represents a novel approach in examining these phenomena. Our model uses A6 cells--a cell line derived from Xenopus collecting duct (CD)--grown on semi-permeable supports. These cells, the standard model of the CD, will be subjected to two experimental conditions: 1. """"""""short-circuiting"""""""" as a model of increased apical Na+ entry; 2. Na+ depletion, where A6 cells are exposed to sodium depleted media, ablating apical Na+ entry. Preliminary data has already revealed changes in the expression of ENaC and Na/K-ATPase subunits when subjected to these different conditions. We hope to characterize the effects of apical sodium entry on the transcription, translation, and post- translational regulation of these channels using biochemical, electrophysiologic, and fluorescent microscopic techniques. In this way, we hope to derive a better understanding of the molecular basis for such entities as diuretic resistance and obstructive uropathy--clinical correlates for our model.
