Dr. Mary Lee Ledbetter's previous work has used simple assays to measure the ability of cultured fibroblastic or epithelial cells to share small cytoplasmic molecules through gap junctions. This cooperation can be inhibited by treatment of the cells with the sodium-pump inhibitor ouabain. When kidney-derived epithelial cells are treated with the steroid hormone aldosterone, their ouabain inhibition is reduced. Recent work has suggested that calcium changes mediate the effects of ouabain and that aldosterone's action may involve an unusual mechanism independent of the cytoplasmic mineralocorticoid receptor. Tests will be carried out to determine whether ouabain and aldosterone also modify the expression an/or distribution of connexins, the gap junction protein, in cooperating cells. Experimental approaches include: connexin protein localization by immunofluorescent staining in intact cells; expression of connexin - specific RNA and protein and protein electrophoretic mobility in cell extracts; and rapid changes in functional communication, measured by dye injection. These studies will not only provide a more complete basis for understanding the interaction between ouabain, aldosterone and cell communication, they will also provide improved tools to study structure-function relationships in gap junction communication for future work. Interactive activities include participation in the core course for first-year graduate students at The Scripps Research Institute. This involves leading discussion sections, designing and implementing laboratory experiences, and offering occasional lectures in the course.
