title: Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.
abstract: Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.
authors.affiliation: Department of Medicine, University of California, San Francisco 94143-0532.
authors.forename: K
authors.initials: K
authors.lastname: Fushimi
authors.forename: J A
authors.initials: JA
authors.lastname: Dix
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008560:Membrane Fluidity; D008954:Models, Biological; D011817:Rabbits