Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume.
OBJECTIVE
Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells.


METHODS
An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume.


RESULTS
AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001).


CONCLUSIONS
In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo.
Stamer
W D
WD
Department of Ophthalmology, The University of Arizona, 655 North Alvernon Way, Tucson, AZ 85711-1824, USA. dstamer@eyes.arizona.edu
Peppel
K
K
O'Donnell
M E
ME
Roberts
B C
BC
Wu
F
F
Epstein
D L
DL
D000260:Adenoviruses, Human / Q000235:genetics; D000293:Adolescent; D000368:Aged; D051398:Aquaporin 1; D020346:Aquaporins / Q000502:physiology*; D001789:Blood Group Antigens; D002463:Cell Membrane Permeability; D048429:Cell Size / Q000502:physiology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D015151:Immunoblotting; D007231:Infant, Newborn; D008875:Middle Aged; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000166:cytology* / Q000378:metabolism
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051398:Aquaporin 1; D012313:RNA