Aquaporin-dependent water permeation at the mouse ocular surface: in vivo microfluorimetric measurements in cornea and conjunctiva.
OBJECTIVE
Fluorescence methods were developed to quantify membrane and tissue water permeabilities at the ocular surface and to compare water transport in wild-type mice versus transgenic mice lacking each of the water channels, aquaporin (AQP)-1, -3, and -5, normally expressed in cornea or conjunctiva.


METHODS
Membrane water permeabilities (P(f)(mem)) of calcein-stained surface epithelial cells were measured from the kinetics of fluorescence quenching in response to rapid (<0.2 seconds) changes in extraocular fluid osmolarity. Tissue water permeabilities (P(f)(tiss)) across intact cornea and conjunctiva--the relevant parameters describing water movement into the hyperosmolar tear film in vivo--were determined by a dye-dilution method from the fluorescence of Texas red-dextran in an anisosmolar solution in a microchamber at the ocular surface.


RESULTS
Osmotic equilibration occurred with an exponential time constant (tau) of 1.3 +/- 0.2 seconds (P(f)(mem) = 0.045 cm/s) in calcein-loaded corneal epithelial cells of wild-type mice, slowing 2.1 +/- 0.4-fold in AQP5-deficient mice; tau was 2.4 +/- 0.1 seconds in conjunctiva (P(f)(mem) = 0.025 cm/s), slowing 3.6 +/- 0.7-fold in AQP3-deficient mice. In dye-dilution experiments, P(f)(tiss) of cornea was 0.0017 cm/s and decreased by greater than fivefold in AQP5-deficient mice. P(f)(tiss) in AQP5-null mice was restored to 0.0015 cm/s after removal of the epithelium. P(f)(tiss) of conjunctiva was 0.0011 cm/s and was not sensitive to AQP3 deletion.


CONCLUSIONS
These results define for the first time the water-transporting properties of the two principal ocular surface barriers in vivo. The permeability data were incorporated into a mathematical model of tear film osmolarity, providing insights into the pathophysiology of dry eye disorders.
Levin
Marc H
MH
Department of Medicine and Physiology, Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94143-0521, USA.
Verkman
A S
AS
D000818:Animals; D051398:Aquaporin 1; D051400:Aquaporin 3; D051402:Aquaporin 5; D020346:Aquaporins / Q000172:deficiency / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D003315:Cornea / Q000033:anatomy & histology / Q000166:cytology / Q000378:metabolism*; D003592:Cytophotometry; D008565:Membrane Proteins / Q000172:deficiency / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009994:Osmolar Concentration; D010539:Permeability; D013666:Tears / Q000378:metabolism; D014867:Water / Q000378:metabolism*
C496021:Aqp1 protein, mouse; C496028:Aqp3 protein, mouse; C496038:Aqp5 protein, mouse; D051402:Aquaporin 5; D020346:Aquaporins; D008565:Membrane Proteins; D014867:Water; D051398:Aquaporin 1; D051400:Aquaporin 3