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Biophys J, April 2001, p. 1783-1790, Vol. 80, No. 4

Cell Volume Kinetics of Adherent Epithelial Cells Measured by Laser Scanning Reflection Microscopy: Determination of Water Permeability Changes of Renal Principal Cells

Kenan Maric,* Burkhard Wiesner,* Dorothea Lorenz,* Enno Klussmann,* Thomas Betz,dagger and Walter Rosenthal*Dagger

 *Forschungsinstitut für Molekulare Pharmakologie, D-10315 Berlin,  dagger Carl Zeiss, Mikroskopie, Vertrieb Berlin, D-10787 Berlin, and  Dagger Freie Universität Berlin, Institut für Pharmakologie, D-14195 Berlin, Germany

The water channel aquaporin-2 (AQP2), a key component of the antidiuretic machinery in the kidney, is rapidly regulated by the antidiuretic hormone vasopressin. The hormone exerts its action by inducing a translocation of AQP2 from intracellular vesicles to the cell membrane. This step requires the elevation of intracellular cyclic AMP. We describe here a new method, laser scanning reflection microscopy (LSRM), suitable for determining cellular osmotic water permeability coefficient changes in primary cultured inner medullary collecting duct (IMCD) cells. The recording of vertical-reflection-mode x-z-scan section areas of unstained, living IMCD cells proved useful and valid for the investigation of osmotic water permeability changes. The time-dependent increases of reflection-mode x-z-scan section areas of swelling cells were fitted to a single-exponential equation. The analysis of the time constants of these processes indicates a twofold increase in osmotic water permeability of IMCD cells after treatment of the cells both with forskolin, a cyclic AMP-elevating agent, and with Clostridium difficile toxin B, an inhibitor of Rho proteins that leads to depolymerization of F-actin-containing stress fibers. This indicates that both agents lead to the functional insertion of AQP2 into the cell membrane. Thus, we have established a new functional assay for the study of the regulation of the water permeability at the cellular level.

Biophys J, April 2001, p. 1783-1790, Vol. 80, No. 4
© 2001 by the Biophysical Society   0006-3495/01/04/1783/08  $2.00


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