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Am J Physiol Cell Physiol 286: C426-C432, 2004. First published October 22, 2003; doi:10.1152/ajpcell.00298.2003
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method

Eugen Solenov,1 Hiroyuki Watanabe,2 Geoffrey T. Manley,2 and A. S. Verkman1

1Departments of Medicine and Physiology, Cardiovascular Research Institute, and 2Department of Neurosurgery, University of California, San Francisco, California 94143-0521

Submitted 14 July 2003 ; accepted in final form 15 October 2003

A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible ~12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in <200 ms in response to addition of cytosol in vitro, indicating that the fluorescence signal arises from changes in cytosol concentration. In astrocytes from wild-type CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with a half-time of 0.92 ± 0.05 s at 23°C, corresponding to an osmotic water permeability (Pf) of ~0.05 cm/s. Pf was reduced 7.1-fold in astrocytes from AQP-4-deficient mice. Temperature dependence studies indicated an increased Arrhenius activation energy for water transport in AQP-4-deficient astrocytes (11.3 ± 0.5 vs. 5.5 ± 0.4 kcal/mol). Our studies establish a calcein quenching method for measurement of cell membrane water permeability and indicate that AQP-4 provides the principal route for water transport in astrocytes.

water transport; aquaporin-4; fluorescence quenching; brain swelling; cerebral edema


Address for reprint requests and other correspondence: A. S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, Univ. of California, San Francisco, San Francisco, CA 94143-0521 (E-mail: verkman{at}itsa.ucsf.edu); http://www.ucsf.edu/verklab.




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