Ion Transport and Communication in Cultured Mammalian Cells

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; R o o t E n t r y F C o m p O b j b W o r d D o c u m e n t 4 O b j e c t P o o l 1 2 3 4 5 6 7 8 9 : ; F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; 9406646 Ledbetter Dr. 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. Furosemide, a modulator of Na+,K+, 2C1- cotransport, stimulates communication. When kidney-derived epithelial cells are treated with the steriod 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 classical Type I mineralocorticoid receptor. Dr. Ledbetter plans to pursue these findings as examples of interactions between regulatory mechanisms that effect ion transport and those that influence junction-mediated communication. She will carry out these studies in her own laboratories as well as in collaboration with Dr. Norton B. Gilula at the Scripps Research Institute (during her sabbatical leave) and with Dr. Fredic S. Fay at the Univ ersity of Massachusetts School of Medicine. The goal is to establish how these and other modulators of ion support influence cell communication. She will use Dr. Gilula s collection of connexin-specific antibodies and nucleic acid probes, to study. a. connexin protein localization by immunofluorescent staining intact cells; b. expression of connexin-specific RNA and protein and protein electrophoretic mobility in cell extracts; and c. rapid changes in functional communication, measured by dye coupling of cells. The PI will also use digital imaging microscopy and spectrofluorimetry of cells loaded with ion-sensitive indicator dyes, along with radioactive tracer experiments to correlate effects on communication with influx, efflux and steady-state content of calcium and potassium and cytoplasmic ph. %%% The results should help to refine new modes for the interaction between ion-transport regulatory mechanisms and regulation of gap-junction-mediated communication. *** ; S u m m a r y I n f o r m a t i o n ( 0 Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT 9406646 Shirley Parker Shirley Parker @ @ @ @ F # Microsoft Word 6.0 2 ; e = e F 4 F j j j j j j j x 1 : Z T 7 x j x j j j j ~ j j j j r 9406646 Ledbetter Dr. 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. Furosemide, a modulator of Na+,K+, 2C1- cotransport, stimulates communication. When kidney-derived epithelial cells are treated with the steriod hormone aldosterone, their ouabain inhibition is reduced. Recent work has suggested that calcium changes mediate the effec