Water permeability in AQP1-expressing Xenopus oocytes when controlling both extracellular and intracellular environments. Experimental and mathematical modeling approaches

DORR RICARDO; OZU MARCELO; CHARA OSVALDO; TORIANO ROXANA; PARISI MARIO

Montevideo

Congreso; 5th Southern Cone Biophysics Congress. 6th International Conference of Biological Physics.; 2007

IUPAP - SAB

The classical method to measure the osmotic water permeability (Pf, cm.s-1) of the cellular membrane using whole Xenopus oocytes (WO)does not allow a direct test on the internal side of the cell and hence in the cytoplasmic side of the water channel. But a recently describedtechnique allows controlling the composition and hydrostatic pressure on both sides of the cellular membrane of emptied-out Xenopusoocytes (EOO) (Ozu et al, J. Biochem. Biophys. Methods (2005) 63:187-200).Results on the osmotic properties of the cellular membrane employing the EOO technique are now presented offering measurements inaquaporin-1 (AQP-1) expressing oocytes.Experiments using WO were used to validate data obtained with the EOO technique. The expected large increase in osmotic permeabilitywas observed in AQP-1 expressing cells, both in control and emptied-out oocytes. Additionally, the AQP-1 inhibitor Mercury chloride wastested on the extracellular but also on the intracellular side of membrane in AQP-1 expressing EOOs, showing asymmetrical behavior.A software model devoted to simulate the time evolution as a function of water and solute permeabilities was developed to analyze the timecourse of the volume variation under an osmotic challenge. Measurements far forwards 50-sec. of the relative volume vs. time curve alloweddiscriminating between two populations in high permeability EOOs. This qualification depended on the necessary time to explode after ahypotonic challenge, and short response (SR) and long response (LR) oocytes were identified.From the mathematical model simulations and fitting with the experimental curves, it can be concluded that only water moved during the first50-sec. after the osmotic challenge in high permeability EOOs. Part of the tested oocytes developed solute permeability under longerexposures to an osmotic gradient. These results extend the utility of the presented experimental approach.