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1 From the Departments of Ophthalmology and 2 Pharmacology, The University of Arizona, Tucson; the 4 Departments of Ophthalmology and 3 Medicine, Duke University, Durham, North Carolina; and the 5 Department of Human Physiology, University of California, Davis.
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Abstract |
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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 [14C]-sucrose/urea equilibration. Permeability of TM monolayers to [14C]-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 [14C]-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.
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Introduction |
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In the human eye, AQP1 is present in many tissues that require the efficient movement of water, including the corneal endothelium, the lens epithelium, the iris epithelium, the nonpigmented ciliary epithelium, and the cells of the conventional outflow pathway.9 10 The conventional outflow pathway contains two cell types, trabecular meshwork (TM) and Schlemm’s canal (SC) endothelial cells, both of which express AQP1.9 10 11 TM cells cover collagen lamellae that form the maze of passages through which water must flow in the outflow pathway. As aqueous humor drains from the eye, it first passes through the tortuous TM until finally reaching and crossing a monolayer of endothelial cells that line SC. Because aqueous humor appears to move primarily by bulk flow around TM cells and not through them (i.e., paracellular rather than transcellular),12 previous studies indicate that intracellular volume of TM cells appears to be a determinant of outflow facility.13 14 15 For example, agents that decrease TM-cell volume increase outflow facility, and agents that increase TM-cell volume decrease outflow facility. These findings suggest that intracellular volume of TM cells in vivo may dictate outflow resistance by affecting the dimensions or direction of the human outflow pathway; particularly in the juxtacanalicular region, where spaces between TM cells approach 1 µm. Whether AQP1 plays a physiological role in outflow across TM cells is unknown.
Defining the functional contribution of AQP1 to the permeability of TM tissue and to outflow facility may be important for understanding the pathologic course of glaucoma, the second leading cause of blindness in the United States.16 Glaucoma is an optic neuropathy that is generally characterized by elevated intraocular pressure resulting from impaired outflow facility. Unknown cellular defect(s) in the TM decrease outflow facility, resulting in an elevation in intraocular pressure that compresses nerve axons in the retina and results in blindness.17 18
The present study was conducted as an initial investigation of the role of AQP1 in TM cells. Using adenovirus (AV)-mediated gene transfer of AQP1 cDNAs, we were able to control the expression of AQP1 in TM cells, thus demonstrating the utility of AQP1 AV as an important tool for studying AQP1 function in these cells. In the present study, we show that altering the expression of AQP1 influenced resting volumes of TM cells and consequently their paracellular permeability. In addition, these studies revealed that knockdown of native AQP1 in TM cells by AQP1 antisense AV decreased native AQP1 protein and correspondingly decreased TM cell volume. Taken together, these data suggest that AQP1 protein levels influence the set point for resting volume in TM cells.
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Methods |
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AV Construction
The AV backbone for the AQP1 sense (s) and antisense
(as) AV constructs was a replication-deficient first-generation AV with
deletions of the E1 and E3 genes.20
This empty AV contains the cytomegalovirus (CMV) promoter and bovine
growth hormone polyadenylation (bGH) site separated by a polylinker
containing a unique XbaI restriction site. A large-scale
preparation of empty AV DNA (described later) was digested with
proteinase K (Sigma, St. Louis, MO) in the presence of 0.5% SDS at
55°C for 2 hours, followed by phenol-chloroform (1:1, vol/vol)
extraction and ethanol precipitation. Viral DNA was then restricted
with XbaI overnight, and the large fragment containing the
bGH and AV (map units 9.3-100) was gel purified from a 0.6% agarose
gel. This DNA fragment served as the right end of both AQP1(s) AV
constructs.
The left ends of AQP1 recombinant viruses were constructed uniquely (see Fig. 1 ). For AQP1(s) AV, a plasmid containing the coding sequence for AQP1, pCHIPev,21 was digested with KpnI and BamHI (giving a 1162-bp fragment) and subcloned into the shuttle vector pSKAC, creating pSKAC/AQP1. pSKAC contains the left end cassette (map units 0.0-1.3) of the AV that is flanked by two unique restriction sites, PmeI and XbaI. The cassette includes the left terminal repeat of AV, a CMV promoter, an AMV translation enhancer, and a polylinker region.
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Approximately 100 ng of appropriate gel-purified left-end fragment of AQP1(s) AV or AQP1(as) AV was ligated to approximately 1 µg of the right-end fragment overnight at 16°C, using T4 ligase (Gibco BRL, Gaithersburg, MD). The ligation mixture was transfected into single 60-mm dishes of strain 293 human embryonic kidney cells (293 cells) using lipofectamine reagent (Gibco). Transfected cells were allowed to lyse without an agar overlay. Cell lysates were subjected to three rounds of freezing-thawing using a dry ice-ethanol bath. Individual viruses were isolated from cell lysates by two consecutive rounds of plaque purification, using an agar overlay.
After isolation, individual viruses were amplified in EBNA-293 cells
(293 cells constitutively expressing EBNA-1 protein from Epstein-Barr
virus; Invitrogen, San Diego, CA). EBNA 293 cells on forty 150-mm
plates (at 
50% confluence) were infected with appropriate AV at a
multiplicity of infection (MOI) of 3. When the majority of cells were
floating (36–48 hours after infection), the cells were harvested by
gentle scraping and collected by a 5-minute centrifugation at
1000g. The cell pellet was resuspended in 20 mM Tris-HCl and
2 mM EDTA (pH 7.4) and the cells were homogenized with 20 strokes
(Dounce homogenizer; Kontes Glass, Vineland, NJ). RNase A was added to
100 µg/ml, and the homogenate was incubated at 22°C for 5 minutes.
Nuclei were removed by centrifugation at 2500g for 10
minutes. CsCl was added to the supernatant to 0.3 g/ml and the
supernatant layered atop a CsCl step gradient (1.3 and 1.4 g/ml in
virus storage buffer [VSB]: 137 mM NaCl, 20 mM Tris-HCl, 5 mM KCl,
and 1 mM MgCl2 [pH 7.4]) and centrifuged for 2
hours at 30,000 rpm (TH64 rotor; Sorvall, Newtown, CT). The virus band
that formed at the 1.3- to 1.4-g/ml interface was removed with a
16-gauge needle and layered atop a 2-ml bed of Sepharose
(Amersham Pharmacia Biotech, Piscataway, NJ) and centrifuged for 2
minutes at 1000 rpm in a tabletop centrifuge (Beckman Coulter,
Fullerton, CA). This step was repeated once more, and the virus
concentration was adjusted to 1 x 1011
plaque forming units (PFU)/ml in VSB. Sucrose was added to 10% (final
concentration) and the virus preparation was stored in aliquots at
-80°C. Each aliquot was used a maximum of two times and discarded.
Individual AV DNA titers were determined by three methods: (1) plaque titration on 293 cells, (2) immunofluorescence microscopy of AV protein expression (anti-penton group antigen, clone 143; Biodesign, Saco, ME) in 293 cells infected with serial dilutions of AV, and (3) absorbance (A) at 260 nm (plaque forming units per milliliter = A260 x dilution x 1010). Infection of cells with AV is expressed as MOI, indicating number of infective virus particles per cell. A range of titers was used to functionally test novel AQP1 AV constructs and was dependent on the specific requirements of a particular assay.
Immunoblot Analysis
SDS-solubilized whole-cell lysates or proteins isolated by cell
surface biotinylation containing 5% ß-mercaptoethanol were
electrophoresed into 12% polyacrylamide gels containing 0.1% SDS.
Fractionated proteins were blotted onto nitrocellulose using a
commercial system, according to the manufacturer’s instructions
(Transblot; Biorad, Hercules, CA). The blots were preincubated for 30
minutes at 22°C in Tris-buffered saline containing 5% nonfat
powdered milk and 0.2% Tween-20 (TBS-T), and were then probed with
affinity-purified anti-AQP1 IgG (1:5000) or anti-ß-actin IgG (AC-15,
1:2000) for 2 hours at 22°C. The blots were washed (three times for
15 minutes each) in TBS-T and were incubated for 2 hours with
horseradish peroxidase–conjugated secondary antibodies (goat
anti-rabbit, 1:5000; Pierce, Rockford, IL). The blots were washed
(three times for 15 minutes each) in TBS-T, and specific labeling was
visualized after enhanced chemiluminescence (Pierce) and exposure to
film (ECL-Hyperfilm; Amersham, Arlington Heights, IL). Immunoblots were
digitized using a gel documentation system, and densitometry was
performed on computer (LabWorks software; UVP, Upland, CA).
Cell Surface Biotinylation
Monolayers of TM cells in 10-cm culture plates were infected
with AQP1(s) AV (MOI, 1.0). Three days after infection, cells were
rinsed with cation-free phosphate-buffered saline (CF/PBS; three times,
10 ml) and incubated with versene for 5 minutes at 4°C.
Cells were rinsed twice with ice-cold biotinylation buffer (100 mM
NaCl, 50 mM NaHCO3 [pH 8.0]) and incubated with
NHS LC-biotin (300 mg/ml, Pierce) in biotinylation buffer at 4°C with
rocking. After 1 hour, cells were rinsed with PBS (three times, 10 ml)
at 22°C and incubated in quenching buffer (100 mM glycine, 25 mM
Tris/HCl [pH 7.4]) for 5 minutes. Cells were again rinsed with PBS
(two times, 10 ml) and solubilized in 2 ml of 0.1 N NaOH. Cell lysates
were diluted 1:10 in PBS and centrifuged at 3000g, and
supernatants were incubated with streptavidin-agarose beads (Pierce) at
22°C with rotation. After 1 hour, beads were washed with 30 bed
volumes of PBS. Proteins specifically bound to beads were extracted by
boiling in buffer containing 3% SDS.
Total RNA Isolation
Ten milliliters guanidine isothiocyanate buffer (4 M guanidine
isothiocyanate, 25 mM sodium acetate, and 650 mM ß-mercaptoethanol)
was added to a 15-cm culture dish confluent with 293 cells at 4°C for
30 seconds. The solution was drawn up and down into a syringe with a
23-gauge needle three times, applied to 12.5 ml of 5.7 M cesium
chloride solution in polyallomer tubes (Beckman) and centrifuged for 24
hours at 25,000 rpm at 20°C using an ultracentrifuge (model L565;
with an SW28 rotor; Beckman). The liquid was removed by
aspiration from the centrifuge tube, leaving a clear pellet. The RNA
pellet was dissolved in diethyl pyrocarbonate–treated water and
ethanol precipitated as described previously.11
The
integrity of the RNA was verified after separation by electrophoresis
into 0.8% agarose gel and visualization using ethidium bromide.
Reverse Transcription–Polymerase Chain Reaction
With AMV reverse transcriptase (Boehringer-Mannheim,
Indianapolis, IN), DNA copies of total RNA or AQP1 antisense RNA from
293 cells were made using oligo dT primers or a primer specific for
AQP1(as) RNA transcripts (5'-CGCGAATTCCTATTTGGGCTTCATCTC-3'),
respectively, as described previously.11
The presence of
AQP1(as) RNA in infected 293 cells was determined using a primer set
specific for AQP1 DNA. The primer set corresponded to nucleotides
316-336 (5'-ATCGGATCCACCGCCATCCTC-3') and 646-671
(5'-ACAGAATTCTATCCCCCGATGAATGG-3') of human AQP1. Amplification of RT
cDNA by Taq polymerase was performed as previously
described,11
using 25 PCR cycles (94°C for 30 seconds,
50°C for 30 seconds, and 72°C for 1.5 minutes). PCR products were
analyzed on 1.4% agarose gels.
Osmotic Challenge Assay
Water permeability was measured as the net fluid movement driven
by an osmotic gradient across AV-infected Madin-Darby canine kidney
(MDCK) cells. Cells were seeded onto filters (Transwell; Costar,
Cambridge, MA; 1 cm2, 0.4-µm pore size) at a
density of 1.5 x 105 cells per well. Cells
were maintained in humidified air containing 5%
CO2 for 2 weeks to allow for cell–cell junctions
to mature. Cells were infected at the apical surface with AV containing
AQP1 in the sense or antisense orientation or with empty AV at an MOI
of 10. Five days after infection, medium was removed from both upper
and lower chambers. Exactly 1 ml fresh, prewarmed isotonic medium (300
mOsM) was added to the lower chamber, and exactly 175 µg hypertonic
medium (400 mOsM) was added to the upper chamber. After 1 hour of
incubation at 37°C, medium from the upper chamber was removed and
volume measured using an analytical balance.
Light-Scattering Measurements
Similar to methods described previously, changes in intensity of
forward light scattering from cell monolayers were used as an indicator
to assess changes in cell volume in real time.22
23
24
In
pilot experiments using both hypertonic and hypotonic challenges,
optimal conditions for resolving volume changes for confluent TM cell
monolayers were determined empirically. TM cells were seeded onto glass
coverslips (13 x 37 x 1 mm) and grown to approximately 80%
confluence. Cells were infected with AV (MOI, 0.1–1.0) and grown for 3
days. Confluence of cell monolayers was verified by phase-contrast
microscopy before assay (IM-35 microscope; Carl Zeiss, Thornwood, NY).
Coverslips containing cells were placed across the diagonal in a 3-ml
polystyrene cuvette fitted with inlet and outlet lines. The cuvette was
perfused at 3 ml/min with Hanks’ balanced salt solution supplemented
with 25 mM HEPES (H-HBSS, pH 7.4), and maintained at 37°C. Once a
stable baseline was obtained (typically, 10 minutes), medium osmolarity
was changed by stopping perfusion and rapidly exchanging the contents
of the cuvette with 9 ml prewarmed hypertonic medium (450 mOsM), using
a 20-ml syringe. Control exchanges of prewarmed isotonic medium were
performed in all cases before hypertonic challenge. Light (512 nm) was
directed toward cell monolayers at a 45° angle of incidence, and
light scattered forward from the monolayer (45°) was monitored in
real time at a wavelength of 520 nm (RatioMaster Spectrofluorometer;
PTI, South Brunswick, NJ). Light beam intersected 1.11
mm2 of the cell monolayer (approximately 300
cells). Comparisons of monolayer permeabilities were obtained on
computer (SigmaPlot software; SPSS, Chicago, IL), by fitting quadratic
polynomials to scatter versus time curves (dV/dT)
for initial response periods (10 seconds) for each condition.
Intracellular Volume Measurements
TM-cell volume was measured by two methods. The volume of
individual TM cells in suspension was determined by electronic cell
sizing using a cell counter (Beckman Coulter, Fullerton, CA), as
described previously.25
For TM cells attached to tissue
culture plates, cell volume was determined by radioisotopic evaluation
of intracellular water space, by using
[14C]-urea and
[14C]-sucrose as markers of total and
extracellular water space, respectively.25
To determine cell volume by electronic cell-sizing methods, mean cell volumes of TM cells in suspension were analyzed by cell counter (model ZM, with a C-1000 Channelizer; Beckman Coulter). The orifice diameter used for these experiments was 140 µm. Cells on T75 culture flasks were infected with appropriate AV (MOI, 0.1) when 80% to 90% confluent. After 4 days, cells were briefly trypsinized and rinsed free of trypsin with PBS that contained a trypsin inhibitor (Clonetics Corp., San Diego, CA). Cells were resuspended at a concentration of approximately 2 x 105 cells/ml in HEPES-buffered minimal essential medium (MEM). Cells were diluted to a final concentration of approximately 50,000 cells/ml, and mean cell volumes of cells in suspension were calculated, by using a minimum of 5000 cells per measurement x three independent measurements for a single data point. Absolute cell volumes (picoliters per cell) were calculated from distribution curves of cell diameter, using a standard curve generated by polystyrene latex beads of known diameter (9.87 and 14.51 µm). Cell suspensions were maintained at 37°C throughout the assay period.
For the radioisotopic method, cell monolayers were cultured in 12-well plates until 80% to 90% confluent and were infected with the appropriate AV (MOI, 0.1). After 4 days, cells were equilibrated for 10 minutes with HEPES-buffered MEM at 37°C in a water bath. The medium was changed to an assay medium that contained either [14C]-sucrose or [14C]-urea (final concentration, 1.0 µCi/ml) in HEPES-buffered MEM. After a 20-minute incubation, the medium was aspirated and each well was rapidly rinsed with ice-cold PBS (four times, 2 ml). Wells were air dried and extracted with 0.1 N NaOH for quantitation of radioactivity and protein. Specific activities (counts per minute per milliliter) of [14C]-sucrose or [14C]-urea in the assay medium were determined and used to calculate water space associated with trapped radioactivity (expressed as microliters per milligram protein). Intracellular volume was calculated as the difference between water space determined for [14C]-urea (a measure of intracellular plus trapped extracellular volume) and water space determined for [14C]-sucrose (a measure of trapped extracellular volume).
Permeability Measurements
Similar to that described previously,35
flux of
[14C]-sucrose across confluent TM cell
monolayers was used to assess paracellular permeability. Cells were
seeded onto filters (Transwell; Costar) at a density of 1 x
105 cells per well, were maintained for 1 day in
culture, and then were infected with AV at an MOI of 0.1. Five days
after infection, cells were assayed. Medium was aspirated from upper
and lower chambers, and exactly 1.0 ml of fresh isotonic medium was
added to the lower chamber. To the upper chamber, exactly 200 µl of
fresh isotonic medium containing [14C]-sucrose
(1 µCi/ml) was added. Cells were maintained at 37°C with rotary
shaking during the incubation period. After 10 minutes 100 µl medium
from the lower chamber was removed, added to 5 ml of scintillation
cocktail, and counted. In control experiments, hypotonic medium (200
mOsM) was used in place of isotonic medium in both chambers.
Statistical Analyses
Experimental results were analyzed by a two-tailed Student’s
t-test assuming unequal variance. The criterion for
significance in all cases was P < 0.05.
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Results |
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Recombinant AQP1 as a Functional Water Channel
TM cell monolayers were infected with AQP1(s) AV (MOI, 1.0) and
tested for the functional presence of recombinant AQP1 protein at their
plasma membrane. To determine whether recombinant AQP1 was at the
plasma membrane of TM cells, cell surface proteins were biotinylated,
solubilized, and purified using streptavidin-agarose. We found
recombinant AQP1 amid biotinylated cell surface proteins (Fig. 3
, lane 4).
To determine whether recombinant AQP1 functions as a water channel in TM cells, cells were infected with empty or AQP1(s) AV, exposed to hypertonic medium (450 mOsM) and cell volume then assessed by means of a forward light-scatter assay. We predicted that an increase in AQP1 expression should increase the permeability of the TM-cell plasma membrane to water and thus alter the rate of initial cell volume changes as cells were exposed to anisosmotic media. By this method, volume changes corresponded to changes in forward light scatter from TM cell monolayers over time. As predicted, TM cells infected with AQP1(s) AV shrank more rapidly in response to a hypertonic challenge (100 mOsM gradient; Fig. 4A ) than cells infected with empty virus or uninfected cells (not shown). Cell volume decreased 1.8- or 4.0-fold (MOI, 0.1 or 0.5, respectively) more rapidly than control. Comparisons were obtained by fitting quadratic polynomials to scatter versus time curves (dV/dT) of initial response (first 10 seconds) to hypertonic challenge.
In addition, we tested the function of recombinant AQP1 in a model system that measures channel-mediated transcellular movement of fluid across MDCK-cell monolayers.27 MDCK-cell monolayers infected with AQP1(s) AV were five times more permeable to fluid flux driven by a 100-mOsM osmotic gradient than cells infected with empty AV (P < 0.001; Fig. 4B ). MDCK cells were used in these experiments, because they form tight cell–cell junctions that limit paracellular fluid flux.
Expression of AQP1(as) AV RNA
The ability of AQP1(as) AV to direct the expression of AQP1(as)
RNA was assessed by RT-PCR in HEK293 cells, which do not express native
AQP1. DNA copies of HEK293 mRNAs were made using reverse transcriptase
and oligo dT primers or primers specific for AQP1(as) mRNA. Analysis of
cDNAs verified the presence of AQP1(as) mRNA in AQP1(as) AV–infected
cells but not in cells infected with empty virus. A primer set spanning
two of AQP1’s three introns amplified DNA of appropriate size (355
bp). PCR products were obtained using either oligo dT or
antisense-specific, oligonucleotide-primed cDNAs as templates (Fig. 5A)
. The ability of AV-driven AQP1(as) RNA to hybridize to native AQP1
mRNA and inhibit expression of AQP1 protein was investigated in TM
cells, which are known to express native AQP1. TM cells were infected
with AQP1(as) AV (MOI, 10) and grown for 5 days. The presence of native
AQP1 in empty AV and AQP1(as) AV–infected cells was determined by
immunoblot analysis. Steady state levels of native AQP1 protein in
cells infected with AQP1(as) AV were decreased by 71.7% ± 5.5%
(mean ± SEM) of control (Fig. 4B)
. Whereas native AQP1 expression
varied between TM-cell strain and passage number, the knockdown level
was very consistent. Comparisons were based on changes in the
nonglycosylated 28-kDa AQP1 species and normalized to ß-actin
protein expression. A representative blot is shown (Fig. 5B
, bottom).
Similar levels of knockdown were obtained using lower concentrations of
virus (Fig. 6)
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AQP1 Protein Expression and Resting Cell Volume
While measuring effects of AQP1 expression on intracellular volume
responses to anisotonic challenges, we observed a novel role for AQP1.
Cellular resting volume of TM cells both in suspension and in monolayer
correlated with AQP1 expression. For these experiments, resting volume
of TM cells was determined using two different methods. Intracellular
volume of individual cells in suspension was resolved by electronic
cell sizing using a cell counter (Coulter), and intracellular volume of
cell monolayers was determined by a urea-versus-sucrose equilibration
assay. Similar to findings by others,27
pilot experiments
in our laboratory showed that specific effects of AQP1 AVs on
resting-cell volume was resolved optimally using low MOIs (< 1.0),
primarily because of nonspecific effects of AV on cell volume that were
noted at high MOIs (>1.0). Therefore, cells were infected with
AQP1(s), AQP1(as), or empty AV at an MOI of 0.1 and grown for 4 days.
Shown in Figure 6
is the protein expression pattern and specifically
AQP1 expression levels in TM cells infected with AV at and MOI of 0.1.
This figure illustrates the typical expression observed for AQP1 in
AQP1(s) and AQP1(as)-infected TM cells. Native AQP1 expression levels
in empty-AV–infected TM cells, however, varied and was dependent on
the primary TM cell line used and its number of cell doublings at the
time of use (see Fig. 5B
).
As shown in Figure 7 , cells infected with AQP1(s) AV had significantly greater mean intracellular volume than cells infected with either empty AV (8.7%, P < 0.05) or AQP1(as) AV (16.5%, P < 0.01). Cells infected with AQP1(as) AV had significantly less mean intracellular volume than cells infected with empty AV (7.8%, P < 0.05). Shown are combined data from four independent experiments using three different TM cell strains. A minimum of 15,000 cells was analyzed for each data point in each experiment.
The effects of AQP1 protein expression were also tested in monolayers of TM cells. Monolayers of TM cells were grown until 80% to 90% confluent and were infected at an MOI of 0.1. After 4 days, cells were assayed for intracellular water volume using a marker for intracellular plus extracellular water, [14C]-urea, and a marker for trapped extracellular water, [14C]-sucrose. Results shown in Figure 8 were similar to those obtained with individual cells. Thus, cells infected with AQP1(s) AV had a significantly greater mean intracellular volume than did cells infected with empty AV (19.9%, P < 0.05) or AQP1(as) AV (28.9%, P < 0.01). Conversely, cells infected with AQP1(as) AV had a smaller, but not significant, mean intracellular volume than empty-AV–infected cells (9.0%, P = 0.18). Retrospectively, we determined that low native AQP1 expression in three of six experiments affected our ability to reach significance.
Effect of AQP1 Protein Expression on Paracellular Permeability
Previous studies have demonstrated that resting volume of TM cells
affects paracellular permeability.35
To functionally
determine the effect of AQP1’s expression on the paracellular
permeability of TM cells, we measured the flux of a paracellular
marker, [14C]-sucrose, across AV-infected TM
cell monolayers grown on permeable filters. Figure 9
shows that TM cells infected with AQP1(s) AV were significantly less
permeable to [14C]-sucrose than
empty-AV–infected (8.0% ± 1.4%, P < 0.001) or
antisense-infected (5.8% ± 1.6%, P < 0.01) cells.
Conversely, the permeability of cell monolayers infected with AQP1(as)
was not significantly different from cell monolayers infected with
empty AV. Although AQP1(as) successfully knocked down low endogenous
AQP1 (see Fig. 6
for example), this assay was not able to resolve
differences between empty and AQP1(as) AV–infected cells. In control
experiments, cell monolayers infected with AQP1(as) or empty AV were
exposed to hypotonic medium (100 mOsM) and assayed for paracellular
permeability. Similar to AQP1(s) AV–infected cells, cells assayed in
hypotonic medium were significantly less permeable to
[14C]-sucrose (due to cell swelling) than cells
in isotonic medium (not shown).
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Discussion |
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In general, resting cell volume is a function of the coordinated activities and expression of a variety of channels and transporters that control the influx and efflux of ions and water at the plasma membrane. The mechanisms whereby cells sense their volume and maintain intracellular volume at a given set point are not fully understood. However, a number of ion transporters and channels are known to participate in the regulation of intracellular volume of a variety of cell and tissue types.29 30 These include ion flux pathways that tend to increase cell volume, such as the Na-K-2Cl cotransporter, the Na/H exchanger coupled to Cl/HCO3 exchange, and the Na/K pump. Volume regulatory pathways also include those ion flux pathways that act to decrease intracellular volume, such as K-Cl cotransport, K channels, and Cl channels. With regard to Cl channels, it is noteworthy that the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel involved in cell volume regulation, affects ion conductance of at least three different ion channels (Na, K, and Cl) and a water channel, AQP3.26 31 32 33 34 In airway epithelial cells, water transport through the AQP3 water channel is functionally coupled to CFTR.33 Thus, AQP1 may influence the activity of an ion channel and/or transporter and affect resting cell volume in TM cells.
Except for the recent characterization of Na-K-Cl cotransporter in TM cells, the specific participants that determine TM resting volume are not known.35 Using bumetanide and ethacrynic acid as inhibitors, the Na-K-Cl cotransporter was shown to contribute significantly to the regulation of TM-cell volume. The functional relevance to outflow cell volume of Na-K-Cl activity in the outflow pathway was detected in one study, but not in another.13 36 Although Gabelt et al.36 failed to observe effects of bumetanide on outflow facility, Al-Aswad et al.13 found that treatment of calf and human anterior chambers in organ culture with bumetanide (10 µM) decreased TM-cell volume by 10% and increased outflow facility by an average of 23%. Thus, small changes in TM-cell volume produced significant changes in outflow facility. This relationship between outflow cell volume and outflow facility is consistent with studies from two other laboratories using outflow perfusion models.14 15 Accordingly, conditions that increase TM-cell volume decrease outflow facility, whereas those that decrease cell volume increase outflow facility. In the present study, AQP1 expression increased cell volume 8.7% to 19.9% (depending on the assay) resulting in decreased paracellular permeability of cell monolayers to sucrose by 8.0%. We propose that a coordinated change in the intracellular volume of TM cells influences outflow facility by modifying extracellular pathways for aqueous flow in the juxtacanalicular region of the outflow pathway. Such a mechanism may account for the nonuniform flow of aqueous through the TM that has been predicted by previous models and may contribute to increased outflow resistance predicted by Johnson’s funneling hypothesis.37 38
A second possibility is that functional effects of outflow cell volume changes occur only at the level of the inner wall of Schlemm’s canal (IWSC), cells that also express AQP1.9 10 Thus, the intracellular volume of IWSC cells may dictate a preferential paracellular or transcellular route for aqueous across the inner wall. Although IWSC cells are held together by tight junctions, a paracellular pathway between IWSC cells was revealed in experiments in which enucleated eyes were perfused with cationic ferritin.40 Thus, shrinkage of IWSC cells may weaken the cell–cell contacts and provide a paracellular route for aqueous humor. Evidence for this idea was first presented in work using vascular endothelial cells. Both in vivo and in vitro studies show that the intracellular volume of vascular endothelial cells modulates water and solute flux across a monolayer of endothelial cells.41 42 43 The cells of the IWSC are functionally similar to vascular endothelium, in that they both present highly regulated barriers to water and solute flux. An alternative possibility must be considered. The enhanced permeability of the IWSC to water may simply be a function of the transcellular movement of water through AQP1 in the plasma membrane of SC cells. Such a mechanism is certainly operational in cell barriers of other tissues that express AQP1.44 45 Thus, the functional role for AQP1 in SC may oppose that in TM, but must be determined experimentally.
Using AQP1(s) AV and AQP1(as) AV to control AQP1 expression, the functional contribution of AQP1 to aqueous outflow facility can now be tested in systems that model the human outflow pathway. For example, infection of TM and SC cells in human anterior chambers in organ culture or in live primates will enable the examination of AQP1’s specific contribution to outflow facility. Further, the specific participation of AQP1 to outflow facility at the level of IWSC can be assessed using cultured SC cell monolayers on permeable supports.28 Such experiments are compelling, because information about channel-mediated (AQP1) versus non–channel–mediated (endocytic) fluid movement across SC can be measured directly.
In addition to providing a valuable tool to understand aqueous outflow function, AQP1 AV may have therapeutic utility. The viability of adenoviral transfer of AQP1 to tissues with a secretory or absorptive deficit has been recently demonstrated.46 After radiation treatment, secretory function was restored to cells of the rat salivary glands using adenoviral delivery of recombinant AQP1 in vivo. These studies were extended in vitro to show that delivery of low virus titers of AQP1(s) AV (similar to those used in the present study) to salivary cell monolayers increased their permeability to water 10-fold.27 Implications of these results for patients with glaucoma who have depressed aqueous outflow function are encouraging but will depend ultimately on the net effect of AQP1 expression in TM and SC cells on outflow facility.
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Acknowledgements |
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Footnotes |
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Submitted for publication November 20, 2000; revised February 21, 2001; accepted March 19, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: W. Daniel Stamer, University of Arizona, 655 North Alvernon Way, Suite 108, Tucson, AZ 85711-1824. dstamer{at}eyes.arizona.edu
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