Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore.
Quantitative determination of rapid water and solute transport and solute reflection coefficients by light-scattering methods is complicated by dependence of vesicle or cell light scattering on nonvolume factors including solution refractive index, cell motion, and membrane aggregation. To overcome these difficulties, a fluorescence technique has been developed to measure accurately (1) osmotic water permeability (Pf), (2) solute permeability (Ps), and (3) solute reflection coefficient (sigma). The time course of vesicle volume is determined by the self-quenching of entrapped fluorescein sulfonate (FS), the best of a series of dyes screened for self-quenching, brightness, and vesicle loading/trapping. To validate the method, rabbit renal brush border vesicles (BBV) were loaded with 1-10 mM FS for 12 h at 4 degrees C and washed to remove extravesicular FS. FS leakage occurred over greater than 6 h at 4 degrees C and greater than 30 min at 23 degrees C. FS fluorescence vs vesicle volume was calibrated from the time course of fluorescence decrease (excitation 470 nm, emission greater than 515 nm) in response to a series of inward osmotic gradients in a stopped-flow apparatus. At 23 degrees C Pf was 0.005 +/- 0.001 cm/s, independent of osmotic gradient size, and inhibited 67% by 0.5 mM HgCl2. Urea Ps was 2 x 10(-6) cm/s with sigma 0.95-1.00 on the basis of the fluorescence time course analysis and the extravesicular [urea] required to obtain zero initial volume flow (null method) when vesicles were loaded with sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
P Y
PY
Department of Medicine, University of California, San Francisco 94143.
Pearce
D
D
Verkman
A S
AS
D000818:Animals; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal; D008871:Microvilli; D009995:Osmosis; D010539:Permeability; D011817:Rabbits; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014508:Urea; D014867:Water
D005456:Fluorescent Dyes; D012996:Solutions; D014867:Water; D014508:Urea