Figure 5.
Inhibition of glycerol permeability. A, addition of extracellular glycerol causes a rapid scattered light intensity (SLI) increase, indicative of water exit, mainly through hAQP1 and hAPQ3, followed by a slower scattered light intensity decrease, indicative of swelling induced by the osmotic influx of water, following glycerol entry mainly through AQP3. Representative example traces show clear inhibition of erythrocyte water and glycerol permeability by addition of 25 μm DFP00173 compared with solvent (1% DMSO). B, relative potency of erythrocyte glycerol permeability inhibition, measured as t½ of the scattered light intensity decrease. Apparent IC50 values were ∼0.2 μm (DFP00173) and ∼0.6 μm (Z433927330). Substance was identified as a significant source for variation in two-way ANOVA at p < 0.0001; n = 6. C, example traces of glycerol permeability in calcein-loaded CHO-mAQP3 cells in an isotonic shrinking assay. DFP00173-treated (25 μm) and solvent control (1% DMSO) cell traces of relative fluorescence intensity are shown. Cells were preincubated in 500 mm glycerol buffer before addition of 500 mm membrane-impermeable sucrose. Fluorescence quenching indicates cellular glycerol efflux along its gradient. D, dose–response curves of glycerol permeability inhibition in CHO cells expressing mAQP3 and mAQP7, respectively. Fluorescence quenching kinetics expressed as t½ were measured. Apparent IC50 values were ∼0.7 μm for DFP00173 (mAQP3), N.D. for DFP00173 (mAQP7), ∼11.5 μm for Z433927330 (mAQP3), and ∼2.6 μm for Z433927330 (mAQP7). n = 3. AU, arbitrary units. Error bars in B and D indicate standard deviation.