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Effect of AQP1 Expression Level on CO₂ Permeability in Bovine Corneal Endothelium

¹ From the School of Optometry, Indiana University, Bloomington, Indiana; and the ² Department of Ophthalmology, University of Arizona, Tucson, Arizona.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
PURPOSE. Corneal endothelial fluid transport is dependent on HCO₃^- and CO₂ fluxes. CO₂ permeability (PCO₂) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO₂. An AQP1 knockout mouse model, however, showed no evidence for CO₂ transport through AQP1 in erythrocytes or lung. Because HCO₃^- and CO₂ fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO₂.

METHODS. BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1.

RESULTS. Expression of AQP1 and osmotic water permeability (control P_f = 0.046 ± 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P_f. Adenoviral empty virus (EV) infection did not change AQP1 expression or P_f. PCO₂ was determined by measuring the rate of intracellular pH decrease after exposure to CO₂/HCO₃^--rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO₂ of BCEC (0.0036 ± 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P_f could also be reduced more than 50% by 3 to 5 minutes’ exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease.

CONCLUSIONS. AQP1 does not contribute to PCO₂ in corneal endothelial cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of the water channel protein aquaporin-1 (AQP1) confers significant increases in membrane osmotic water permeability (P_f; see¹ for review). AQP1 and other aquaporins are often highly expressed in epithelial cells involved in large amounts of osmotically coupled water transport (e.g., the kidney² ) and active salt pumping (e.g., salivary gland³ ) and AQP1 knockout mice⁴ show significant reductions in P_f and tissue water fluxes. In contrast, absence of aquaporins in lung alveoli reduces P_f but has no effect on isotonic fluid transport, indicating that epithelia involved in small levels of fluid transport may not require the high P_f provided by aquaporins.^{5 6} Furthermore, aquaporins can be highly expressed in cell types with no known water transport function.⁷ Thus, there may be other undiscovered functions of membrane aquaporin expression.

Small solutes are excluded from AQP1; however, recent reports indicate that AQP1 may be permeable to CO₂. CO₂ permeability (PCO₂) was increased approximately 40% in oocytes expressing AQP1.^{8 9} Further, proteoliposomes reconstituted with AQP1 were reported to have four times higher PCO_2.¹⁰ These reports have suggested that increased PCO₂ may be particularly advantageous for those cells involved in rapid CO₂ exchange, such as erythrocytes, lung alveoli, renal proximal tubule, choroid plexus, and ciliary epithelium. However, a recent study using AQP1 knockout mice showed no significant change in erythrocyte or alveolar PCO₂ and did not demonstrate an effect of AQP1 on PCO₂ in proteoliposomes.¹¹

Using an antisense transfection approach to alter AQP1 expression, we examined whether AQP1 is permeable to CO₂ in confluent cultures of bovine corneal endothelial cells (BCECs). AQP1, but not AQP2 through 5,¹² is highly expressed in the corneal endothelium^{13 14} and confers a relatively high P_f. Ion-coupled fluid transport by the corneal endothelium maintains corneal hydration and transparency, but at 6 µl/cm² per hour, its secretion is at a relatively low level.^{15 16} Endothelial fluid transport is dependent on the presence of HCO₃^- and is significantly slowed by carbonic anhydrase inhibitors.^{15 16 17 18 19} In addition to strong expression of cytosolic carbonic anhydrase (CAII),²⁰ corneal endothelium also expresses the membrane-bound carbonic anhydrase (CAIV) on its surface.²¹ A recent model of HCO₃^- transport across the apical membrane of corneal endothelium includes CO₂ flux from intracellular to extracellular compartments with conversion of CO₂ to HCO₃^- catalyzed by CAIV.²² Thus, if AQP1 were permeable to CO₂, it could have an important role in vectorial transport of HCO₃^- across the corneal endothelium. In this study, we show that reduction in endogenous AQP1 expression or overexpression of AQP1 significantly alters osmotic water permeability in cultured BCEC but has no effect on PCO₂.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Bovine corneal endothelial cells (BCECs) were cultured to confluence as previously described.²³ Briefly, primary cultures from fresh cow eyes were established in T-25 flasks with 3 ml of Dulbecco’s modified eagle’s medium (DMEM) and 10% bovine calf serum gassed with 5% CO₂ and 95% air at 37°C and fed every 2 to 3 days. Primary cultures were subcultured to three T-25 flasks and grown to confluence in 5 to 7 days. The resultant second-passage cultures were then further subcultured onto coverslips and used for the experiments described herein.

Antisense Oligonucleotides
Antisense oligodeoxynucleotides (AOs), 5'-CAGCCCTCCAGAAGAGCTTCTTCTT-3', complementary to bases +16 to +40 of AQP1 mRNA were used to reduce expression in cultured BCECs. As a control, sense oligodeoxynucleotides (SOs; +14 to +38, 5'-TCAAGAAGAAGCTCTTCTGGAGGGC-3') were also used. Computer searches of GenBank and EMBL (Vector NTI ver 5.2 software; InforMax; North Bethesda, MD) showed no significant homologies to other sequences. The first nine bases from the 5' end and the penultimate seven bases from the 3'-end of both oligonucleotides were phosphorothioated (Gibco Life Technology, Grand Island, NY). In preparation for immunoblot analysis, cells were grown in six-well plates. For physiological measurements cells were grown on glass coverslips. When BCEC cultures were 60% to 80% confluent (1 day after seeding), 5 µg/ml oligonucleotides was applied in complete culture medium. Culture media were changed on days 3 and 5 and fresh oligonucleotides were added. On day 7, cells were collected for protein extraction. Cell-coated coverslips were used for measurement of osmotic water P_f and separate coverslips for measurement of PCO₂.

AQP1 Adenoviruses
The adenovirus (AdV) backbone for the AQP1 sense and antisense AdV constructs was a replication-deficient first-generation AdV with deletions of the E1 and E3 genes.²⁴ This empty AdV (EV) contains the cytomegalovirus (CMV) promoter and bovine growth hormone polyadenylation (bHG) site separated by a polylinker that was used to clone AQP1 DNA, as described previously.²⁵

Two AQP1 recombinant viruses were constructed by a unique method. For AdV sense-AQP1 (SV), a plasmid containing the coding sequence for AQP1, pCHIPev,²⁶ was digested and subcloned into the shuttle vector pSKAC, creating pSKAC/AQP1. pSKAC contains map units 0.0 to 1.3 of the AdV, which includes the left terminal repeat of AdV, a CMV promoter, an AMV translation enhancer, and a polylinker region. For AdV antisense-AQP1 (AV), an 807-bp DNA fragment encoding antisense AQP1 was generated by polymerase chain reaction using pCHIPev as a template and primers that delineated the full coding sequence of AQP1, as described previously.²⁵ The DNA fragment was subcloned into pSKAC, creating pSKAC/AQP1as. DNA fragments containing AQP1 sense or antisense DNA were liberated from pSKAC after restriction and ligated into adenovirus, as described previously.²⁵ Human embryonic kidney cells (strain 293) were transfected with ligation mixture, and individual viruses were isolated from cell lysates by two consecutive rounds of plaque purification using an agar overlay, as described previously.²⁵ Individual viruses were amplified in the 293 cells and purified over cesium step gradient. Individual AdV DNA titers were determined by three methods: 1) plaque titration on 293 cells, 2) immunofluorescence microscopy of AdV protein expression (anti-penton group antigen, clone 143; Biodesign, Kennebunk, ME) in 293 cells infected with serial dilutions of AdV; and 3) absorbance at 260 nm (plaque-forming units [pfu]/ml = A₂₆₀ x dilution x 10¹⁰).

Adenoviral Infection of BCECs
The three adenoviruses EV, AV, and SV were used to infect BCECs at a multiplicity of infection of 10. Cultured BCECs grown to 80% to 90% confluence on six-well tissue culture plates or coverslips were exposed to virus in complete culture media. On days 3 and 5, culture media (without virus) were replenished. On day 7, the cells from six-well plates were collected for extraction of protein to perform Western blot analysis. Coverslips were used for osmotic water P_f or PCO₂.

Immunoblot Analysis
Cultured BCECs were dissolved directly in sample buffer, sonicated (model 250 sonicator; Branson, Danbury, CT) briefly on ice, and centrifuged at 6000g for 5 to 10 minutes. An aliquot of the supernatant was taken for protein assay using the Lowry method (Bio-Rad, Richmond, CA). ß-Mercaptoethanol (5%) and bromphenol blue were added to the remainder of the supernatant. The samples were boiled for 5 minutes and applied to a 10% polyacrylamide gel with a 4.5% stacking gel. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane overnight at 4°C. Membranes were incubated in phosphate-buffered saline (PBS) containing 5% nonfat dry milk for 1 hour at room temperature. The blots were then incubated with rabbit polyclonal AQP1 antibody (1:200; Alpha Diagnostics; San Antonio, TX), in PBS containing 5% nonfat dry milk for 1 hour at room temperature with shaking. Next, the blots were washed five times for 5 minutes each with PBS/Tween-20, incubated with goat anti-rabbit secondary antibody coupled to horseradish peroxidase (Sigma, St. Louis, MO) for 1 hour at room temperature, washed with PBS/Tween-20 five times for 5 minutes each, and developed by enhanced chemiluminescence (ECL). Films were scanned to produce digital images, and the density of the bands was estimated using Un-Scan-It software (Silk Scientific, Orem, UT).

Determination of Osmotic P_f
Relative changes in cell volume of confluent BCECs cultured on glass coverslips were determined by a fluorescence-quenching technique.²⁷ Briefly, collisional quenching of a halide sensitive quinolinium fluorescent dye by intracellular quenchers is reduced during cell swelling and increased during cell shrinkage. Strict volume sensitivity is obtained by perfusion in the absence of halides.²⁷ Cells were loaded with 6-methoxy-N-ethylquinolinium (MEQ) by exposure to 10 µM 6-methoxy-N-ethyl-1,2-dihydroquinoline (DiH-MEQ; Molecular Probes, Eugene, OR)²⁸ for 10 minutes followed by 20 minutes’ washing at room temperature. Coverslips (25 mm in diameter) were clamped into a microscope perfusion chamber that was modified from that previously described.²³ Briefly, the chamber formed a perfusion slot (3.5 x 16 x 1.5 mm deep) with the clamped coverslip at the bottom and the slot left open at the top. One end of the slot was fit with 23-gauge stainless steel tubing connected to PharMed tubing (Fisher Scientific; Fairlawn, NJ). Ringer solution was delivered to the chamber periodically for 7 seconds through glass syringes at 0.5 ml/sec and rapidly removed at the opposite end of the perfusion slot by suction. Cells were viewed with a x40 (0.75 numeric aperture) objective (Zeiss, Thornwood, NY) on the stage of an inverted microscope (Diaphot; Nikon, Melville, NY). Fluorescence excitation (365 ± 10 nm) and data collection (50 sec^-1) were obtained using a DeltaRam PTI fluorescence system controlled by Felix software (Photon Technology International, Monmouth Junction, NJ). The composition of the halide-free Ringer solution used was (in millimolar): 150 Na⁺, 4 K⁺, 0.6 Mg²⁺, 1.4 Ca²⁺, 146 NO₃^-, 1 HPO₄^-, 10 HEPES^-, 2 gluconate^-, and 5 glucose, with pH adjusted to 7.50 at 22°C. Osmolality was adjusted to 300 mOsm/kg with sucrose. Hypotonic solutions were obtained by 33% to 50% dilution with nanopure water.

After exposure to hypotonic solutions, fluorescence increased and reached a new steady state within 20 seconds. These data were smoothed (Savitzky–Golay), and the initial slope (dF/dt) determined using Felix analysis software. P_f was calculated by

(1)

where P_f is the osmotic water permeability coefficient (in centimeters per second), dF/dt is the initial slope of fluorescence change, F_I is the baseline fluorescence level, F_b is the background fluorescence determined at the end of each experiment by complete quenching of MEQ fluorescence with 150 mM thiocyanate, S/V is the surface-to-volume ratio assumed to be 2500 cm for 4-µm-thick confluent cells, V_w is the partial molar volume of water (18 cm³/mole), and C is the difference in osmolality between solutions.

Determination of PCO₂
BCEC PCO₂ was determined by measuring the rate of intracellular pH (pH_i) change after exposure to CO₂. The same perfusion setup as was used for P_f was used for determination of PCO₂. Solutions were Na⁺-free to remove any effect of the Na⁺/HCO₃^- cotransporter or Na⁺/H⁺ exchanger on pH_i.²³ HCO₃^--rich Ringer contained (in millimolar): 80 K⁺, 70 N-methyl-D-glucamine (NMDG⁺), 0.6 Mg²⁺, 1.4 Ca²⁺, 118 Cl^-, 28.5 HCO₃^-, 1 HPO₄^-, 10 HEPES^-, 2 gluconate^-, and 5 glucose, equilibrated with 5% CO₂ and pH adjusted to 7.50 at 22°C. For HCO₃^--free solutions, KHCO₃ was replaced by K-gluconate, and the solution was equilibrated with air. Confluent BCECs cultured on glass coverslips were loaded with the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), by incubation in an Na⁺-free, HCO₃^--free Ringer solution containing 5 µM of the acetoxymethyl ester of BCECF (BCECF-AM; Molecular Probes) at room temperature for 30 to 60 minutes. Dye-loaded cells were then kept in Ringer for at least 30 minutes before use. Fluorescence ratios (495 ± 10 and 440 ± 10 nm excitation) were obtained at 50 sec^-1 and were calibrated against pH_i by the high K⁺-nigericin technique.²⁹ A calibration curve that follows a pH titration equation has been constructed for BCECs.²³

After exposure to the CO₂/HCO₃^- solution, pH_i rapidly decreased (half-life 0.5 seconds) and reached a new steady state in 4 to 5 seconds. These data were smoothed (Savitzky–Golay) and the initial slope (dpH_i/dt) determined using analysis software (Felix). The apparent H⁺ influx rate, JH⁺, due to dissociation of CO₂ and formation of H₂CO₃ is (dpH_i/dt) · ß · V/S, where ß is the intrinsic buffering capacity of BCECs (10 mM/pH²³ ) and V/S is the volume-to-surface ratio. The fraction of CO₂ entering the cell that forms H₂CO₃ is K/(H + K), where H is the intracellular H⁺ concentration (steady state pH_i in the presence of CO₂) and K is the apparent dissociation constant (7.24 x 10^-7). Therefore, CO₂ influx (JCO₂) is JH⁺ · [(H + K)/K],⁹ and

(2)

where [CO₂] is 1.36 mM (0.036 mM/mm Hg x 37.7 mm Hg).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
AQP1 Expression
Antisense oligonucleotides were first used in an attempt to reduce AQP1 expression in cultured BCECs. Oligonucleotide concentrations higher than 10 µg/ml caused an increase in dead floating cells, reduced cell proliferation, and reduced cell density after 7 days of culture compared with control. Cultures exposed to 5 µg/ml of oligonucleotides were morphologically indistinguishable from control cultures. Furthermore, fluorescence levels after MEQ or BCECF loading were in the same range as control cultures, indicating cell viability and membrane integrity. Therefore, in all oligonucleotide experiments reported, 5 µg/ml was used. In pilot experiments (not shown), visual inspection of BCEC cultures after 7-day exposure to Texas red–labeled oligonucleotides indicated that approximately 99% of cells had been transfected. Figure 1A shows an immunoblot for AQP1 after sense and antisense oligonucleotide exposure. To determine the approximate percentage of decrease, the densities of the native band (28 kDa), and the glycosylated band (45 kDa) were summed. In the experiment shown in Figure 1A , antisense exposure reduced AQP1 expression to approximately 60% of control (by densitometry), whereas parallel incubation with sense oligonucleotides produced an approximately 11% reduction. In a separate experiment (not shown), immunoblots indicated approximately 50% antisense oligonucleotide decrease.

View larger version (57K): [in this window] [in a new window]   Figure 1. Immunoblot analysis of BCECs probed with polyclonal AQP1 antibody. (A) Oligodeoxynucletide transfection. Lanes 1 and 2: untreated cells (control); lanes 3 and 4: cells exposed to sense and antisense oligonucleotides for 7 days (7d). (B) Effect of adenoviral transfection with AV, SV, and EV. All lanes (A, B) contained 60 µg of protein.

BCEC Permeability Measurements
Transfected BCECs were first characterized to show that the altered expression in AQP1 was concomitant with P_f. Figure 2A shows representative data of relative volume changes (F - F_b/F_I- F_b) for each condition. In each case, 20 seconds of data are shown, starting at the time when MEQ fluorescence began to increase on exposure to hypotonic solution. A new steady state level of fluorescence was achieved in approximately 10 seconds for SV-infected cells; approximately 15 seconds for control, EV-infected, and SO-transfected cells; and between 25 and 35 seconds for AO-transfected and AV-infected cells. The averaged P_f for each condition is summarized in Figure 2B . AO transfection and AV infection showed 36.5% and 49% reduction in P_f, respectively, relative to control cells (P_f = 0.046 cm/sec). SO-transfected and EV-infected cells had P_f that was not significantly different from control. However, SV infected cells showed a small (37%) but significant increase in P_f relative to EV-infected cells.

View larger version (18K): [in this window] [in a new window]   Figure 2. Pf of BCECs from control and transfected cells measured by MEQ fluorescence quenching. (A) Relative volume (F - Fb/FI - Fb). Data are arranged in descending order of rate of volume change (CON, control). Cells were bathed in 300 mOsm isotonic Ringer. Beginning data indicate exposure to Ringer diluted by 50%. (B) Pf averages ± SE; number of experiments is indicated above each bar; *AO significantly different from CON (P < 0.05); #SV and AV significantly different from EV.

View larger version (25K): [in this window] [in a new window]   Figure 3. PCO2 of cultured BCECs. (A) Control BCECs were loaded with the pH-sensitive fluorescent dye BCECF initially bathed in a CO2/HCO3--free Ringer. Down arrows: Exposure to CO2/HCO3--rich Ringer; up arrows: return to CO2/HCO3--free Ringer. (B) Expanded section of pHi decrease after exposure to CO2 for control (CON) and EV- and AV-transfected cells. Calculated PCO2 (1.85 x 10-3 cm/sec) was similar for all three. (C) PCO2 average ± SE; number of experiments is indicated above each bar.

View larger version (19K): [in this window] [in a new window]   Figure 4. Effects of pCMBS on Pf and CO2 Pf. (A) Cells were loaded with MEQ and the bathing solution was switched from isotonic (300 mOSM) to 33% diluted Ringer, indicated by the hatched boxes. After the control challenge, cells were exposed to 0.5 mM pCMBS for 3 minutes and challenged again with hypotonic Ringer. pCMBS was then removed and cells were bathed in 5 mM DTT for 10 minutes, followed by a 5-minute wash in isotonic Ringer and then challenged with hypotonic Ringer. (B) pHi changes due to CO2 exposure before and after exposure to pCMBS. Down arrows: Exposure to CO2/HCO3--rich Ringer; up arrows: return to CO2/HCO3--free Ringer. At the break in the data, cells were pre-exposed to 0.5 mM pCMBS for 3 minutes. Two subsequent CO2 exposures in the continued presence of pCMBS were similar to control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

AQP1 expression in BCECs was altered using antisense oligonucleotides or CMV-promoted expression of AQP1 sense and antisense mRNA. The oligonucleotide approach was reasonably effective, inhibiting approximately 45% of AQP1 expression and leading to a decrease in P_f of 37%. The adenoviral approach was more effective, inhibiting approximately 60% of AQP1 expression and reducing P_f by 49%. This is a reasonable relation between reduced AQP1 expression and reduced P_f, because P_f includes both AQP1-mediated water flux as well as non-AQP1 mediated flux (e.g., through the naked membrane). This has been demonstrated previously, for example, in heterozygous AQP1 null mice, in which red blood cell AQP1 protein expression was reduced by 20%, and P_f was reduced by approximately 10%.¹¹ By contrast, SV infection increased AQP1 expression more than threefold; however P_f increased by only 37%. AQP1 expression in control BCECs is exclusively at the apical and basolateral plasma membranes.¹⁴ Considering that AQP1 membrane protein density can be very high,¹ it may be that in cells overexpressing AQP1, trafficking to the outer membranes is inefficient. Further, functionality of human AQP1 in bovine cells may be compromised. Regardless, the difference in functional range in AQP1 expression between SV- and AV-infected cells was approximately 2.5-fold.

The calculated P_f of control cells was 0.046 cm/sec, giving a density of approximately 7500 AQP1 monomers/µm². This P_f is slightly higher than that reported for erythrocytes (0.018 cm/sec)¹¹ and airspace-capillary barrier (0.022 cm/sec),⁵ but almost four times lower than that reported in renal proximal tubules (0.15 cm/sec).³¹ The previous estimate of P_f for cultured BCECs by Echevarria et al.¹³ was approximately 0.0093 cm/sec. One significant difference between the studies is that Echevarria et al. used trypsinized BCECs that had settled onto coverslips for 4 hours. Thus, there was not a confluent monolayer. The calculation of P_f by Echevarria et al.¹³ used the entire cell membrane, giving an S/V ratio that is approximately five times greater than that used in the present study. As they suggest, because water would enter the cells primarily through the apical membranes, they may have underestimated P_f. It is remarkable that the differences in P_f are completely explained by the choice of the S/V ratio. Considering that water flux may also occur across lateral membranes through the leaky intercellular junctions, then the current estimate of P_f could be considered the upper limit. Furthermore, our results show that accurate P_f estimates can be obtained with confluent cultures using the fluorescence quenching technique.

PCO₂ in BCECs was measured using the rate of pH_i decrease after exposure to CO₂, as measured by the fluorescence ratio of BCECF. The experiments were performed in the absence of Na⁺, to block any pHi regulatory effects from the Na/HCO₃^- cotransporter or the Na⁺/H⁺ exchanger. Chloride was left in the solutions, because the Cl^-/HCO₃^- exchanger is not expressed in cultured BCECs.³² That pH_i regulation was fully blocked is apparent from Figure 3A , which shows no increase in pH_i over 20 seconds during exposure to CO₂/HCO₃^- solutions. The apparent PCO₂ of cultured confluent BCEC was approximately 0.0036 cm/sec. This value is similar to that measured in Xenopus oocytes expressing AQP1⁹ and approximately 3x less than that measured in erythrocytes using a rapid stopped-flow procedure.¹¹ In comparison with bilayer studies performed in the presence of carbonic anhydrase and high buffering capacity (PCO₂ = 0.35 cm/sec),³³ the values reported for cells are low, which is probably due to unstirred layers, intracellular barriers to diffusion, and/or carbonic anhydrase kinetics.

Given the density of AQP1 monomers estimated for BCECs from the measured P_f, it is possible to predict the cellular PCO₂ from estimates of single-channel PCO₂. The single-channel PCO₂ (2 x 10^-14 cm³/sec)¹¹ for AQP1 estimated from oocyte data⁹ would predict a control PCO₂ in BCECs that is approximately four times that measured. The single-channel AQP1 PCO₂ (5 x 10^-15 cm³/sec)¹¹ estimated from the proteoliposome data of Prasad et al.¹⁰ predicts a PCO₂ of 0.0038 cm/sec in BCECs, similar to that found. However, the approximately 50% decrease in P_f in antisense-treated BCECs should have reduced PCO₂ by 0.0019 cm/sec, a change that would have been easily detected by the current method. On the contrary, using the upper limit on single-channel AQP1 PCO₂ estimated for erythrocytes (3 x 10^-16/cm³),¹¹ a value that suggests little CO₂ transport by AQP1, a 50% decrease in AQP1 expression in BCECs would cause a 0.0001 cm/sec decrease in PCO₂, a change that falls within the SE of our data. This demonstrates that the current findings are consistent with those reported by Yang et al.,¹¹ indicating that AQP1 PCO₂ is insignificant.

In AQP1-transfected oocyte studies, 15 minutes’ exposure to 1 mM pCMBS slowed CO₂ fluxes; however, reversal with a reducing agent was not shown.⁹ In addition, a mercurial insensitive AQP1 mutant that retains water permeability showed increased PCO₂ and no effect of pCMBS.⁹ This indicates that pCMBS was not toxic to the oocytes; however, with BCECs the pCMBS dosage and time used for oocytes would sometimes prove toxic, as evidenced by accelerated losses of BCECF fluorescence. In contrast, 3 to 5 minutes’ exposure to 0.5 mM pCMBS, which reduced P_f by more than 50% and did not reduce PCO₂, was well tolerated by BCECs. Prasad et al.¹⁰ measured increased rates of pH change in proteoliposomes when reconstituted with AQP1, which was eliminated in the presence of 1 mM HgCl₂. Yang et al.¹¹ were unable to measure an effect of AQP1 reconstitution on proteoliposome PCO₂, and their study suggests that HgCl₂ may inhibit carbonic anhydrase.

In summary, antisense oligonucleotide transfection and adenoviral infection of sense and antisense AQP1 led to significant changes in cultured BCEC AQP1 expression and osmotic water permeability. Measurements of PCO₂ in untransfected cells versus transfected cells as well as pCMBS-treated cells, show that AQP1-dependent P_f in BCECs does not significantly affect PCO₂.

	Footnotes

 
Supported by Grant EY08834 from the National Institutes of Health.

Submitted for publication August 9, 2000; revised October 4, 2000; accepted October 16, 2000.

Commercial relationships policy: N.

Corresponding author: Joseph A. Bonanno, Indiana University, School of Optometry, 800 E. Atwater Avenue, Bloomington, IN 47401. jbonanno{at}indiana.edu

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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