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Am J Physiol Cell Physiol (October 22, 2003). doi:10.1152/ajpcell.00298.2003
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Submitted on July 14, 2003
Accepted on October 15, 2003

SEVEN-FOLD REDUCED OSMOTIC WATER PERMEABILITY IN PRIMARY ASTROCYTE CULTURES FROM AQUAPORIN-4 DEFICIENT MICE MEASURED BY A CALCEIN QUENCHING METHOD

Eugene Solenov1, Hiroyuki Watanabe1, Geoffrey T Manley1, and A. S Verkman1*

1 Medicine and Physiology and Neurosurgery, U.C.S.F., San Francisco, CA, USA

* To whom correspondence should be addressed. E-mail: verkman{at}itsa.ucsf.edu.

A calcein fluorescence quenching method was applied to measure osmotic water permeability in primary cultures of brain astrocytes from wildtype and aquaporin-4 (AQP4) deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing 2-fold 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. In astrocytes from wildtype CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with t1/2 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 AQP4-deficient mice. Temperature-dependence studies indicated an increased Arrhenius activation energy for water transport in AQP4-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 AQP4 provides the principal route for water transport in astrocytes.




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