Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport

Kunyan Kuang1, Yansui Li1, Quan Wen1, Zheng Wang2, Jun Li1, Yingqing Yang1, Pavel Iserovich1, Peter S. Reinach2, Janet Sparrow3, Friedrich P. J. Diecke4, and Jorge Fischbarg1,5

1 Departments of Ophthalmology, 5 Physiology and Cellular Biophysics, and 3 Anatomy and Cell Biology, Columbia University, New York 10032; 4 Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103; and 2 Department of Biological Sciences, State University of New York-Optometry, New York, New York 10010


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Although Na+-K+-2Cl- cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [3H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s-1. Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na+-K+-2Cl- cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.

bumetanide binding; epithelial polarization; stromal thickness; specular microscopy; turnover rate; sodium-potassium-two chloride cotransporter


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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CORNEAL ENDOTHELIUM TRANSPORTS fluid from stroma to aqueous humor to maintain constant hydration of the cornea. It is generally assumed that the fluid transport is driven by bicarbonate transport from the basolateral to the apical membrane. However, the specific electrolyte transport mechanisms, which generate the driving force for the fluid transport, and the location of these transport mechanisms in the polarized endothelium are not well understood. Using 86Rb to trace K+ uptake, we have established recently the presence of a bumetanide-sensitive K+ uptake in cultured bovine corneal endothelial cells (CBCEC) (8). This K+ uptake depends on the presence of extracellular Na+ and Cl- and is activated by hypertonic challenge. It thus has the characteristics of a Na+-K+-2Cl- cotransporter (NKCC). Based on our findings we postulated that this protein could contribute up to 30% of the transendothelial electrolyte transport and hence to fluid transport across this preparation. We have recently also obtained evidence for expression of NKCC mRNA in bovine corneal epithelial and endothelial cells (5) as well as in CBCEC, supporting our postulate.

The suggestion that a Cl- transport in parallel to bicarbonate transport might contribute to the transendothelial fluid movement has been challenged. Riley et al. (27) have reported that bumetanide neither affected K+ uptake in dissociated cells of freshly dissected corneal endothelia nor did it affect the rate of deturgescence of corneal endothelium. The authors, therefore, concluded that NKCC may be expressed in cultured cells but not in their native tissues. Similar claims that the expression of the cotransporter is induced in cultured cells but not present in vivo have been made for bovine lens epithelial cells (2), rabbit proximal tubular cells, and porcine aortic endothelial cells and rat smooth muscle cells (25). On the other hand, Western blot analysis has demonstrated the existence of cotransport protein in cultured and freshly dissected bovine aortic endothelial cells and brain microvascular endothelial cells (32). Due to this controversy, we have further investigated the Na+-K+-2Cl- cotransporter of corneal tissue, and we present in this study new details about the NKCC isoform expressed in freshly dissected and cultured corneal endothelial and epithelial cells obtained by RT-PCR methods. In addition we have examined the distribution of the cotransporter between different membrane domains as determined by [3H]bumetanide binding and investigated the effect of bumetanide on the thickness of the rabbit cornea measured by specular microscopy. The data presented lend support to our earlier postulate (8) that Na+-K+-2Cl- cotransport proteins are present in the corneal endothelium and epithelium and contribute significantly to regulation of stromal thickness of the rabbit cornea.


    MATERIALS AND METHODS
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Cell culture. CBCEC cells were prepared according to Narula et al. (22). Briefly, after bovine eyes arrived from a local slaughterhouse, corneas were excised under sterile conditions. The endothelial layer was covered with a 0.05% trypsin-0.53 mM EDTA-Na solution (GIBCO) at 37°C for 5 min. The endothelium was then scraped gently, and the dispersed cells were aspirated and transferred into DMEM (GIBCO) containing 5% fetal bovine serum (FBS), 2 ng/ml human recombinant basic fibroblast growth factor, and 100 U/ml penicillin and 100 ng/ml streptomycin. Bovine corneal epithelial cells (CBCEPC) were obtained and cultured using the methods described by Tao et al. (28). The endothelium and a large part of the stroma were removed by peeling from the excised corneas during microscopic dissection. The thinned corneas were placed epithelial side down in a dish containing serum-free DMEM/F-12 with 5% dispase II at 37°C for 30-40 min. After the dispase medium was replaced with DMEM/F-12 culture medium containing 10% FBS, the loosened epithelial cells were scraped off and further dispersed by passing them though a syringe attached to a 23-gauge needle. The cells were centrifuged and then resuspended with DMEM/F-12 containing 10% FBS, 5 µg/ml insulin, and 40 µg/ml gentamicin. Both CBCEC and CBCEPC cells were grown in 25-cm2 culture flasks, kept in a 5% CO2 humid incubator at 37°C, and fed with fresh culture medium every 2-3 days.

RNA isolation and RT-PCR reaction. Freshly dissected bovine corneal endothelial (FBCE) and epithelial (FBCEP) layers were lysed off their basement membranes with ULTRASPEC RNA reagent (Biotecx). CBCEC and CBCEPC grown to confluence were lysed directly in a culture flask by adding the same reagent and passing the cell lysate several times through a pipette. After extraction of total RNA from FBCE, FBCEP, CBCEC, and CBCEPC, the first strand of cDNA was synthesized from 5 µg of each total RNA using SuperScript RNase H-RT, and a random hexamer primer (SuperScript, GIBCO). Two specific primers used in PCR reaction were based on the sequence of the Na+-K+-2Cl- cotransport protein in bovine aortic endothelial cells [BNKCC1 (32)]. The sense primer (BNKCC1s) corresponded to nucleotides +1726 to +1749, namely, 5'-gggTATCTCAgTAgCCggAATggAg-3', and the antisense primer (BNKCC1a) corresponded to nucleotides +2721 to +2745, 5'-ggAggATCCCCAgTTCACATCTgg-3'. Since these primers are located in different exons (26), the PCR fragment sought would be a product of cDNA and not of genomic DNA amplification. After incubating at 94°C for 5 min to denature the RNA/cDNA hybrid, we lowered the melting point temperature (Tm) in the first five PCR cycles (1 min at 94°C, 2 min at 48°C and 3 min at 72°C) to maximize the annealing. We then increased the Tm in the remaining 35 cycles to enhance the specificity (1 min at 94°C, 2 min at 55°C, and 3 min at 72°C). The PCR product was ligated into pGEM-T Easy Vector (Promega, WI) followed by transformation into JM 109 high efficiency competent cells. Plasmids were then harvested and purified using Wizard Plus Minipreps DNA purification system (Promega). The isolated DNA was sequenced by DNA Facility (Columbia-Presbyterian Cancer Center), and the sequence analysis was done by using an Email FASTA server.

[3H]bumetanide binding. CBCEC were cultured on 25-mm Costar permeable inserts until 1-2 days after confluence. CBCEC confluence was monitored daily by measuring the transendothelial electrical resistance with an Endohm chamber and Epithelia Cell Volt-Ohm meter (EVOM; World Precision Instruments, Tampa, FL) (33).

Bumetanide binding was determined by using a protocol similar to that reported by Haas et al. (15). This procedure includes as a key component the determination of binding to nonspecific sites by exposure to a large (in our case, 30-fold) excess of nonlabeled bumetanide. In brief, the procedure began by transferring a set of inserts for a short time from the original six-well culture dish to a second six-well dish kept on ice. Unless otherwise specified, the apical compartments contained unmodified culture medium. When [3H]bumetanide binding to the basolateral membrane was being determined, the destination wells contained 2 ml of preequilibration culture medium labeled with [3H]bumetanide at either 0.25 µM or 7.5 total (labeled plus unlabeled) bumetanide concentration. This exposure served to equilibrate the label within the extracellular space of the layer, thus preventing the dilution of the [3H]bumetanide in the subsequent (final) incubation medium. At that point, the apical solution was aspirated, and the inserts were immediately transferred to a final incubation six-well dish. These wells contained 1.5 ml of culture medium labeled with [3H]bumetanide at either 0.25 µM or 7.5 total (labeled plus unlabeled) bumetanide concentration (binding medium) prewarmed to 37°C. Prewarmed culture medium was then added to the apical compartment too, and the sample was incubated with gentle shaking for 40 min.

To determine [3H]bumetanide binding to the apical membrane, a set of inserts was once more placed in a six-well dish on ice, which contained 2.0 ml of unlabeled culture medium per well. At that point, the apical solution was aspirated and replaced with 0.6 ml of ice-cold [3H]bumetanide preequilibration medium (once more at either 0.25 or 7.5 µM total bumetanide concentration, as above). After brief exposure, this medium was then aspirated, and the inserts were transferred to a six-well dish containing 1.5 ml of prewarmed (37°C) unlabeled medium per well. At that point, 0.6 ml of prewarmed [3H]bumetanide binding medium (with 0.25 or 7.5 µM unlabeled bumetanide) was added to the apical compartment, and the dish was incubated as described above.

In both cases, a 50-µl aliquot of the binding medium was taken for specific activity determination. After incubation, the apical solutions were removed, and the inserts were washed by immersing them 10 times in 75 ml of ice-cold wash solution (isotonic PBS) in a 100-ml beaker. This washing procedure was repeated twice. The filters were then cut from the inserts with a scalpel, were immersed in liquid scintillation cocktail EcoLite(+) (ICN), and were counted in a Rack Beta 1219 LKB instrument. For the experiments in which hypertonic solutions were used, 100 mM sucrose was added to both apical and basolateral incubation media. The incubation time in such media was also 40 min. In each group of inserts, one or two were used for protein determination by the Lowry method (18). The specific (saturable) and nonspecific binding fractions were obtained by fitting to the data the function
Mass of bound bumetanide 

= {<IT>A</IT> · [bumetanide]&cjs0823;  (<IT>K</IT><SUB>d</SUB> + [bumetanide])} + <IT>B</IT> · [bumetanide]
where A is the maximal mass of saturably bound bumetanide, B is the slope of the nonspecific binding component, and Kd is the dissociation constant of the bumetanide-cotransporter complex. As has been shown, the bumetanide binding constant corresponds closely to its half-maximal inhibitory concentration (11, 13). We have therefore used as Kd the inhibition constant (Ki) value of 51 nM reported earlier by our laboratory (8) for this preparation. From the equation above, we calculate both the saturable and the nonspecific amounts bound at 0.25 µmol/l of bumetanide as well as the maximal amount of saturably bound bumetanide.

Immunocytochemistry. CBCEC were subcultured either on permeable inserts as above or on two-well Lab-Tek chamber slide systems (Nunc, Naperville, IL). Experiments were done 1-2 days after confluence. Layers were washed twice with HEPES-buffered Hanks' balanced salt solution (medium 199; GIBCO BRL) at 37°C, fixed for 30 min in PLP fixative (2% formaldehyde, 75 mM lysine, 10 mM sodium periodate, 45 mM sodium phosphate, pH 7.4) (21), washed in PBS (Dulbecco's phosphate-buffered saline; GIBCO BRL) three times, permeabilized with 0.075% saponin in PBS for 20 min, washed in PBS three times, treated with 1% SDS in K+-free PBS for 5 min, and washed in PBS three times. The fixed and permeabilized cell monolayers were then incubated in blocking solution (15% goat serum, 0.3% Triton X-100, 20 mM sodium phosphate, 0.9 mM NaCl) for 30 min. After the blocking solution was removed by aspiration, the monolayers were exposed to T4 antibody (19) [mouse monoclonal anti-human Na+-K+-2Cl- cotransporter (IgG) plus 5% goat serum, 0.2% BSA, in PBS] at dilutions from 1:1,000 to 1:10,000. The optimal dilution was 1:5,000 under our conditions. Incubation lasted 60 min in a humid chamber, covered and at room temperature. After three washes in PBS and two washes in 5% goat serum in PBS, the monolayers were incubated in Rhodamine Red-X-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:200 for 90 min in the humid chamber, covered and at room temperature. Samples were prepared and kept in the dark to prevent light-induced damage to Rhodamine Red-X.

Nuclear counterstaining and mounting. Samples were equilibrated briefly in 2× SSC solution (300 mM NaCl, 30 mM sodium citrate, pH 7.0 titrated with HCl) and were then incubated for 20 min in 2× SSC solution containing 5 µg/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN). Incubation was terminated with three washes in 2× SSC solution (1 min each), after which samples were incubated for 5 min in Sytox Green (Molecular Probes, Eugene, OR) diluted 1:300 vol/vol in 2× SSC solution, and were then rinsed five times (1 min each) in 2× SSC solution. The upper part of the chamber slide system was then quickly removed, leaving only the cells on the slides. Finally, coverslips were applied to the samples using one drop per well of H-1000 Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The excess mounting medium was aspirated, and the coverslips were secured with clear nail polish.

The preparations were screened for fluorescence with an Axiovert S100 microscope (Carl Zeiss, Thornwood, NY) using excitation wavelengths of 480 and 545 nm to detect emission by nuclear staining (Sytox Green) or by antibody staining (Rhodamine Red-X), respectively. Preparations showing staining were then examined with a scanning confocal microscope (LSM 410; Carl Zeiss). Excitation came from its argon-krypton laser producing lines at 488 or 568 nm. Fluorescence in the xy plane was recorded at different depths, and fluorescence in xz and yz planes was obtained from the combined xy images using Zeiss LSM-PC software. The images were enhanced using Adobe Photoshop software (San Jose, CA).

Measurement of stromal thickness of deepithelialized rabbit cornea. Rabbit corneas were obtained from New Zealand albino rabbits (~2 kg). Animal procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1985). Rabbits were euthanized with an overdose of pentobarbital sodium solution (Butler, Columbus, OH) injected into the marginal ear vein. The eyes were enucleated immediately using the technique of Dikstein and Maurice (9). The deepithelized cornea was mounted in a Dikstein-Maurice chamber held in a thermal jacket kept at 36.5°C and viewed with a specular reflection microscope (9). The endothelial side of the cornea was superfused with solutions at a rate of 1.4 ml/h by a syringe pump (Razel Scientific Instruments, Stamford, CT), while the stromal surface was covered with silicone oil. Stromal thickness was measured in conventional fashion (1) by focusing in sequence on the stroma-oil and endothelium-fluid interfaces and using the microscope micrometer to gauge their separation. Readings were taken every 15 min for 4 h.

Endothelial superfusion. The medium used was a HEPES-HCO<SUB>3</SUB><SUP>−</SUP> Ringer solution containing (in mM) 94.1 NaCl, 37 NaHCO3, 3.8 KCl, 1 KH2PO4, 0.8 MgSO4, 1.7 CaCl2, 6.9 glucose, and 20 HEPES. The pH was 7.4, and the osmolality was 290 mosmol/kgH2O. Ouabain, bumetanide, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and para-bromophenacyl bromide (pBPB) were from Sigma (St. Louis, MO). Stock solutions of the reagents were obtained by dissolving them in DMSO (except for ouabain, which was dissolved in Ringer solution). The final concentration of DMSO in the superfusion medium was 0.1%. The data points in Figs. 3-7 represent the average ± SE of four or more experiments.


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Cloning and sequencing of RT-PCR products. Agarose gel electrophoresis of the PCR products from FBCE, CBCEC, FBCEP, and CBCEPC revealed in each case bands of ~1,000 bp (Fig. 1). This size is consistent with the expected molecular size of NKCC1 PCR-amplified fragments designed from two primers: bNKCC1s (+1726 to +1749) and bNKCC1a (+2721 to +2745). These bands were excised from the gel, inserted into plasmid vectors, and sequenced (Fig. 2). In all cases, we found that the cDNA sequences were nearly identical to that of the bovine aortic endothelial bNKCC1 (GB: BTU70138) (32). In the example shown in Fig. 2, the difference amounted to three bases of 340, or ~ 1%. The sequence referred to corresponds to the isoform present in secretory epithelia and in nonepithelial cells and is 93% identical to the human colon bumetanide-sensitive hNKCC1 (GB: HSU30246), 89% identical to the mouse inner medullary collecting ducting cells mNKCC1 (GB: MMU13174) and rat parotid rtNKCC1 (GB: AF051561), 75% identical to shark sNKCC1 (GB: U05958), and 70% to rabbit kidney rNKCC2 (GB: U07547); this last is an absorptive isoform.


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Fig. 1.   Agarose gel electrophoresis of PCR products amplified with primers bNKCC1s (sense) and bNKCC1a (antisense) that were designed from bNKCC1. Lane 1: 1-kb DNA ladder; lane 2: freshly isolated bovine corneal endothelial cells (FBCE); lane 3: cultured bovine corneal endothelial cells (CBCEC); lane 4: freshly isolated bovine corneal epithelial cells (FBCEP); lane 5: cultured bovine corneal epithelial cells (CBCEPC). A band of ~1,000 bp is seen in lanes 2-5. A DNA band of ~500 bp is seen in lane 6, which corresponds to the RT-PCR product of the control RNA from the GIBCO kit.



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Fig. 2.   DNA and corresponding amino acid sequences of the Na+-K+-2Cl- (NKCC) fragment found. Underlined bases indicate the primers utilized. Bold bases mark places where the sequence here differs from that of Yerby et al. (32); the corresponding residues in the deduced amino acid sequence are boxed. Predicted transmembrane segments are indicated by bold amino acid residues.

[3H]bumetanide binding. CBCEC monolayers reached confluence within 1 wk. The maximal transendothelial specific resistance was attained 1-2 days after confluence and was 55.2 ± 0.8 Omega  × cm2. At that time, [3H]bumetanide (plus either 0.25 or 7.5 µM unlabeled bumetanide) was added to the solutions bathing either the basolateral or apical sides of the inserts. The results are summarized in Fig. 3. The open bars represent the raw data for total binding at a bumetanide concentration of 0.25 µM. The gray bars represent the bumetanide component bound to saturable sites at a bumetanide concentration of 0.25 µM, whereas the solid bars represent the computed total saturable component at infinite bumetanide concentration. The hatched bars represent the bumetanide bound to nonspecific sites at a bumetanide concentration of 0.25 µM.


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Fig. 3.   Sidedness and osmolarity dependence of [3H]bumetanide binding to CBCEC. Open bars, total bound bumetanide bound at 0.25 µM ambient bumetanide concentration ([bumetanide]). Gray bars represent computed saturable fraction (at 0.25 µM ambient [bumetanide]), whereas solid bars represent the computed maximal or total saturable bound fraction. Hatched bars, computed nonspecific binding (at 0.25 µM ambient [bumetanide]). Left: experiment comparing apical vs. basolateral binding; right: separate experiment comparing bumetanide binding to the basolateral membranes in isotonic vs. 33% hypertonic solution. Each experimental point (bar) represents the mean of 4 determinations.

Figure 3 shows two sets of experiments. On the left hand side, the two sets of experimental data serve to compare the amount of apical vs. basolateral binding. As can be seen, saturable binding (whether at 0.25 µM or infinite bumetanide concentration) is vastly higher at the basolateral side. In contrast, nonspecific binding at both locations is not significantly different (P = 0.18, two-tailed t-test).

On the right hand side, the two sets of data illustrate how hypertonicity affects basolateral [3H]bumetanide binding to CBCEC. When both apical and basolateral medium were changed to hypertonic medium (by adding 100 mM sucrose), saturable [3H]bumetanide binding to the basolateral membrane of CBCEC increased (from 1.62 ± 0.14 pmol/mg protein in isotonic medium to 2.74 ± 0.47 pmol/mg protein in hypertonic medium). The increase was significant (P = 0.03, t-test, n = 6, 1 tail).

Immunocytochemistry. Confocal microscopic images showed lateral expression of T4 antibody using Rhodamine Red-X-conjugated goat anti-mouse IgG. Ten to twelve serial optical sections of given samples were obtained in the xy plane; a section from one of the samples plus the three-dimensional (3D) reconstruction for that series is shown in Fig. 4. The images indicated the presence of T4 antibody at the lateral membrane domains, concentrated within 2 µm of the basal membrane (not shown). There is also evidence for basal staining in the 3D reconstruction panels (Fig. 4) and in optical sections (not shown) near the bottom support. There was some cytosolic staining, but its density was much lower than seen at the lateral or basal membranes. No nuclear staining was observed, and no fluorescence at the apical membranes was observed either. Control experiments with mouse IgG substitution for the primary antibody showed no staining.


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Fig. 4.   Three-dimensional reconstruction of confocal images of CBCEC. Red: rhodamine indirectly labeled NKCC1 antibody. Green: Sytox green dye labeling nucleic acids. The large panel (side: 67.7 µm long) represents a cross section of the cell layer. The horizontal and vertical white lines through it denote the planes of the adjoining xz and yz sections, respectively. At the top and right, the xz and yz cross sections shown were obtained from the combined serial optical sections of this cell layer using Zeiss LSM-PC software. Marker atop the large panel: 10 µm. The 3-dimensional reconstruction software artifactually fuses the images of the bottom of the cells with that of the permeable support, creating the yellow lines.

Effects of bumetanide on stromal thickness of deepithelialized rabbit cornea. Under the conditions of our measurements, the deepithelialized stroma is bounded by silicone oil on the outside and the endothelial layer on the inside. Hence, fluid moves in or out of the stroma only across the endothelium, and changes in stromal thickness correspond to transendothelial fluid flows. The normal stroma is partially dehydrated and tends to imbibe water across the endothelium, which is countered by the endothelial fluid transport mechanism. Thus, in this context, stable stromal thickness corresponds to a normal rate of fluid transport, and variations in that rate will induce either shrinking or swelling of the stroma. Our initial experiments were designed to establish 1) optimal conditions for continuous superperfusion and 2) the maximal superperfusion rate for solution change consistent with preparation homeostasis. We found that at a continuous superperfusion rate of 1.4 ml/h with HEPES-HCO<SUB>3</SUB><SUP>−</SUP> solution, the corneal thickness remained nearly constant and the maximal swelling observed over a period of up to 4 h was 3.0 ml/h or less (not shown). Similar results were obtained when TES-HCO<SUB>3</SUB><SUP>−</SUP> buffer was substituted for HEPES-HCO<SUB>3</SUB><SUP>−</SUP> (not shown). We therefore chose this superperfusion rate for control conditions. Next we assessed the maximal superperfusion rates that corneas would tolerate when changing the superperfusion solution. Corneas were superfused with HEPES-HCO<SUB>3</SUB><SUP>−</SUP> Ringer at the control rate of 1.4 ml/h for 30 min, then changed to rates of 50.9, 10.6, and 3.54 ml/h for 5 min, and then returned to the control rate. Changing the rate to 3.54 ml/h did not affect the corneal thickness significantly. However, faster rates did have significant effects and led to increases in corneal thickness (not shown). We decided to use a superperfusion rate of 3.54 ml/h as standard during solution exchanges; at that rate, the chamber volume (0.3 ml) would be replaced in ~5 min.

Since all drugs tested were prepared as stock solutions in DMSO, we also tested the effect of a range of DMSO concentrations on the maintenance of corneal thickness. At 0.1% (vol/vol), DMSO had no significant effect on the maintenance of corneal thickness. However, at 0.2 and 0.4% of DMSO we observed a significant, dose-dependent increase in corneal swelling (not shown). As a consequence, vehicle concentration was kept at 0.1% or less in all relevant experiments. As the control experiments in Fig. 5 show, the stromal thickness was steady for ~1 h and increased steadily afterward at a rate of some 3.2 µm/h.


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Fig. 5.   Effect on stromal thickness of bumetanide at the 4 concentrations indicated compared with perfusion with control solution. Each experimental point is the mean of values obtained from 4 corneal endothelial preparations.

We next investigated the effect of bumetanide on fluid transport. In contrast to what was observed in a previous report (27), bumetanide exposure led to a dose-dependent inhibition of fluid transport (Fig. 5). As can be seen there, the rate of fluid transport apparently stabilizes at a reduced rate in each case, and the corneal thickness tends to a new and higher steady-state value. For each bumetanide concentration, we were able to fit exponential buildup curves to the data (not shown), and thus we calculated their initial slopes or initial rates of swelling. Figure 6 depicts such initial rates of swelling plotted against the relevant bumetanide concentrations; as can be seen, the result is a rectangular hyperbola typical of saturable processes, with half-maximal rate of swelling at ~20 µM bumetanide and maximal rate of swelling of ~22 µm/h. As an interesting sideline, as the inset shows, the bumetanide effect began with a delay, visible during the period between 30 and 60 min, and which depended on the concentration of inhibitor. Such a type of delay is consistent with the fact that the inhibitor, added to the aqueous side, would have to diffuse across the leaky tight junctions to reach the basolateral membrane regions where the cotransporter is located, according to the evidence given here and to the separate evidence recently published (17).


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Fig. 6.   Initial rate of stromal swelling and delay of the effect (inset), both as functions of bumetanide concentration. The data in Fig. 5 were fit to exponential buildup curves, and the initial slopes (plus the errors of the fitted parameters) are shown in the main plot. Data for the insert were also obtained from those in Fig. 5. All fitting was done using the program Origin (Microcal, Northampton, MA). Km, Michaelis-Menten constant; Vm, maximal velocity.

To validate our conditions and put into perspective the findings with bumetanide, we inhibited fluid transport using 1 mM ouabain. Figure 7 shows that this led to a fast swelling (~36 µm/h), and washing with fresh solution after 1 h or more did not reverse the inhibition observed. This is to be compared with the more modest rates of swelling observed with bumetanide (Figs. 5 and 6). The anion channel blockers NPPB and pBPB at a concentration of 100 µM had only slight effects on the maintenance of corneal thickness and caused a swelling of 7.4 ± 1.3 and 6.4 ± 1.6 µm/h, respectively (not shown).


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Fig. 7.   Ouabain-induced increase in stromal thickness. All solutions included 0.1% DMSO. Each experimental point is the mean of values obtained from 4 corneal endothelial preparations. Ctrl, control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our investigation addresses the questions whether the Na+-K+-2Cl- cotransport protein is expressed in corneal endothelial cells and, if expressed, whether it contributes to fluid transport across the corneal endothelium. Previous work in our laboratory (8) established the presence of this transport mechanism in CBCEC. However, other investigators have been unable to observe a bumetanide-sensitive rubidium uptake (27) in endothelial cells of freshly dissected rabbit cornea and have therefore concluded that the cotransporter is not expressed in the native tissue but only in proliferating cells. Although other work in our laboratory has demonstrated the presence of NKCC1 mRNA in freshly dissected corneal endothelia and epithelia as well as in cultured cells (5) of these tissues, that is not definitive proof that the protein is expressed and functional in these epithelia.

Using RT-PCR methods we find that mRNA encoding NKCC1 cotransporter protein is present in cultured corneal endothelial and epithelial cells and, most importantly, in freshly dissected corneal endothelial and epithelial cells. This is the first time this is documented at the molecular level for the corneal endothelium. We have identified an RNA segment encoding 341 amino acid residues of the 1,201-residue bovine NKCC1 sequence described by Yerby et al. (32). In terms of the deduced amino acid sequence, this segment extends from bNKCC1 residue 413 to 753 and includes part of predicted transmembrane helix 4 and helices 5-12. The amino acid sequence of this partial clone is 99% identical to the sequence of bKNCC1 (32). As for the expression of the protein itself, we presently confirm and extend the recent findings of Jelamskii et al. (17); using a monoclonal antibody (T4) against NKCC, we find a basolateral location for the protein in cultured cells (Fig. 4). Our data are consistent with the report by Jelamskii et al. (17), describing immunofluorescent staining of membrane regions of cultured and freshly isolated corneal endothelial cells with a polyclonal antibody (N1) to the Na+-K+-Cl- cotransporter. Therefore, the combined evidence from Jelamskii et al. (17) and ours contradicts the suggestion (27) that this cotransporter is present only in proliferating cells such as cultured cells of corneal limiting epithelia.

The second question addressed by our investigation was the location of the transport sites and their density. Forbush and Palfrey (11) demonstrated first that membranes containing NKCC isoforms bind [3H]bumetanide reversibly with a saturable and nonspecific component. A Scatchard analysis of the saturable binding data was consistent with unimolecular binding kinetics, suggesting the binding of one bumetanide molecule per site (11). As a consequence of these findings, bumetanide binding has been used to document the presence and location of active cotransporter sites and to measure their density. Our binding studies with [3H]bumetanide (Fig. 3) show that the transport site density on the basolateral membrane domain is more than an order of magnitude greater than that of the apical membrane. Moreover, it is possible that the apparent apical binding is actually due to diffusion of some apically applied [3H]bumetanide to the basolateral region and that therefore bumetanide binding is restricted to the basolateral region only. As mentioned above, using immunohistochemistry, a recent paper (17) and our own data locate NKCC in rabbit corneal endothelium to the lateral membranes.

The density of bumetanide binding sites among tissues varies by more than two orders of magnitude (19) and ranges from 0.12 pmol/mg protein in Madin-Darby canine kidney cells (12) to 55 (16) and 85 (30) pmol/mg protein for Ehrlich-Lettre mouse ascites cells and rabbit parotid cells, respectively. We find that CBCEC bind ~1.6 pmol/mg protein (Fig. 3), which is similar to the binding density reported for tracheal and bronchial epithelia (15). This binding site density is significantly increased by some 70% (to ~2.7 pmol/mg protein) in hypertonic solutions (395 mM). This increase in bumetanide binding sites is consistent with the 30% increase in bumetanide-sensitive K+ uptake reported earlier for CBCEC and for the same hypertonicity (8). Whether this increase is due to augmented translocation or activation of the cotransporters remains to be established for this and other systems.

From the density of bumetanide binding sites reported here and the rates of bumetanide-sensitive K+ uptake in this tissue, we can calculate the rate of turnover of the NKCC1 transport proteins. Using values obtained in our laboratory (7, 8), we obtain an average K+ uptake of 75.2 ± 1.3 nmol · mg protein-1 · 10 min-1 or 125.3 ± 2.2 pmol · mg protein-1 · s-1 (n = 56) for the same experimental conditions as used for the binding experiments. At a baseline bumetanide binding site density of 1.6 ± 0.14 pmol/mg protein, the turnover rate is 78.3 ± 7.0 s-1. Moreover, from the ambient electrolyte concentrations used (in mM: [Na+] = 140, [Cl-] = 105, [K+] = 4.8) and the apparent dissociation constants we have obtained [in mM: KNa = 21.1, KCl = 28.1, KK = 1.33, (7)], using the equation ENa2ClK = [Na][Cl]2[K]/(KNaK<SUB>Cl</SUB><SUP>2</SUP>KK + K<SUB>Cl</SUB><SUP>2</SUP>KK[Na] + KClKK[Na][Cl] Kk[Na][Cl]2 + [Na][Cl]2[K]), we can calculate that the transporter is only 73% saturated. Hence, using the ideal maximum velocity (Vmax) value, the turnover is 107 ± 10.7 s-1. Our value is in agreement with the turnover numbers of 74 and 70 K+ · site-1 · s-1 reported for smooth muscle of normotensive and hypertensive Wistar Kyoto rats, respectively (24). However, significantly different turnover numbers of 300 K+ · site-1 · s-1 and 4,000 s-1 have been reported for vascular endothelial cells (23) and duck red blood cells (13), respectively.

From the immunocytochemistry evidence, most of the NKCC is located in the lateral membrane. These results corroborate those recently reported by Bonanno and colleagues (17) for the corneal endothelium using T4 and N1 antibodies; they found immunolabeling along the lateral margins of CBCEC cells (along with some apparent intracellular staining). In addition to the sections given before (17), we show here views in the xz and yz planes (Fig. 4) that evidence definite lateral and some basal staining. In our case, membrane staining with T4 is also visible, whereas, in the prior paper, T4 in particular labeled only a cytoplasmic fraction. The smaller fraction of cytosolic staining may represent either protein traffic to or from the membrane and/or a functional reserve to be activated under certain circumstances.

Transendothelial fluid flow. The rates of change in stromal thickness observed under control conditions (Figs. 5 and 7) are in close agreement with prior determinations in the literature (9), including our own (1). The rate of swelling resulting from ouabain inhibition (~36 µm/h, Fig. 7) is also in line with prior determinations (10, 29). In contrast, as mentioned in RESULTS, the data in Figs. 5 and 6 are at variance with those in the prior paper of Riley et al. (27); in our case, a dose-dependent inhibitory effect of bumetanide on transendothelial fluid flow is clearly visible.

As Figs. 5 and 6 show, both the initial rate of swelling as well as the maximal amount of swelling rise with increasing inhibitor concentration. The asymptotic rate of swelling calculated (some 20 µm/h) is ~50% of that observed with ouabain (37 µm/h, Fig. 7). This value for the rate of swelling in rabbit corneal endothelial preparations can be approximately compared with the rate of electrolyte uptake by the Na+-K+-2Cl- cotransporter observed by us (8) in cultured endothelial cells. If one assumes that such rate of electrolyte uptake contributes to generate isotonic fluid, it would correspond to ~25% of the rate of fluid transport. The current observation that bumetanide acts on rabbit corneal endothelium in vitro and affects corneal thickness is consistent with a NKCC role in cell homeostasis and/or fluid transport, and appears novel in the clarity and consistency of the effects noted (Fig. 5). There was a partial precedent in that, on challenge with 50 µM furosemide, Bonanno and colleagues (17) observed a decrease in the intracellular Cl- concentration in 50% of the CBCEC tested and only in 1/10 of fresh cell preparations examined. Problems with lack of or variable effects of bumetanide have been observed before, and are discussed below.

As Fig. 5 shows, the concentrations of bumetanide required to induce swelling range from 20 to 100 µM, or approximately three orders of magnitude higher than the the Ki determined (8) for bumetanide-sensitive K+ uptake by CBCEC. While we have no explanation for this difference, we note that a recent communication by Alvarez et al. (3) on in vitro rabbit lens reports the presence of a NKCC isoform by immunofluorescence labeling but also a lack of inhibitory effect of bumetanide, which they ascribe to either inactivity of the transporter or inaccessibility to the drug. As the insert in Fig. 6 shows, the onset of swelling of the corneal stroma in response to different bumetanide concentration is delayed, and the duration of the delay is inversely related to the inhibitor concentration. Since only the apical region of the in vitro cornea is being superfused, the delay may correspond to 1) the time required for the inhibitor to diffuse across the apical junctions to the basolateral membrane region and 2) the time required for its concentration to build up in the local environment of the NKCC binding site. For a molecule of the size of bumetanide (mol wt approx  364), we calculate a diffusion coefficient of ~4.5 × 10-6 cm2/s. However, the presence of the endothelium entails an additional restriction to diffusion from aqueous to stroma [Dfree solution/Drestricted approx  1/2,000 (20)]. From these numbers and the observed delays between addition of bumetanide and the beginning of its effects, junctional diffusion can significantly retard the buildup of bumetanide necessary for it to reach an inhibitory level at the site of action. In addition, while the Ki for NKCC1 inhibition in other tissues is <2 µM, in the present case some 20 µM were required for significant inhibition (Fig. 6), a concentration not far removed from the maximal ones tested (100 µM). Such restrictions to the buildup of bumetanide are in themselves significant; they are consistent with the results of Alvarez et al. (3), with the ones reported here, and with those of Riley et al. (27). In this last paper, moreover, bumetanide was dissolved directly into water, and this factor might have contributed to the negative results they reported.

Similar restrictions would apply to the diffusion of ouabain. However, in that case, the minimal inhibitory concentration would be of the order of 10-5 M (4). Since we are using it at 1 mM concentration, the inhibitory concentration will be reached much faster than in the case of bumetanide.

Further examination of the data yields what seems an interesting compensatory process by the endothelium. In Fig. 8 we compare the rate of swelling induced by ouabain with that due to 100 µM bumetanide. As can be seen there, the bumetanide inhibitory effect seems to decrease as a function of time. If one calculates the difference between the initial rate of swelling and the following values of that rate, the result is given by the curve labeled "compensatory" in Fig. 8. It would appear that during the 3-h interval recorded, the inhibition of NKCC triggered the progressive increase of another component underlying fluid transport, leading to partial reversal of the bumetanide-induced inhibition. A logical candidate for such upregulation is the Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporter known to be present in corneal endothelium (6, 31), but the precise mechanism involved remains to be determined.


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Fig. 8.   The graph compares the stromal rates of swelling obtained with 1 mM ouabain and 100 µM bumetanide. For the case of ouabain, a linear fit was obtained. For bumetanide, the initial rate was computed from en exponential buildup fit to the data points.

We have also investigated Cl- channel inhibitors to assess whether Cl- channels participate in the fluid transport mechanism. These inhibitors lead to some swelling, but only to an extent (not shown) that is less than one-half of that induced by bumetanide. The mechanism by which the NKCC contributes to fluid transport remains to be determined. On the one hand, we know that a sizable amount of Cl- goes into the cells basolaterally via the NKCC. On the other hand, we still do not know fully where and by which mechanisms it leaves the cell.


    ACKNOWLEDGEMENTS

We acknowledge technical advice from Dr. C. Lytle.


    FOOTNOTES

This work was supported by National Eye Institute Grant EY-06178 and by Research to Prevent Blindness. The [3H]bumetanide utilized was a generous gift of Dr. Mark Haas. The T4 monoclonal antibody developed by Drs. Christian Lytle and Bliss Forbush III was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.

Address for reprint requests and other correspondence: J. Fischbarg, Departments of Physiology & Cellular Biophysics, College of Physicians and Surgeons, Columbia Univ., 630 West 168th St., New York, NY 10032 (E-mail: jf20{at}columbia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 June 2000; accepted in final form 2 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akiyama, R, Kuang K, Chiaradia P, Roberts CW, and Fischbarg J. Effects of acetylcholine, carbachol and mannitol on rabbit corneal endothelial function as assessed by corneal deturgescence. Graefes Arch Clin Exp Ophthalmol 235: 384-387, 1997[ISI][Medline].

2.   Alvarez, LJ, and Candia OA. Na+-Cl--K+ cotransport activity in cultured bovine lens epithelial cells and its absence in intact bovine lenses. Exp Eye Res 58: 479-490, 1994[ISI][Medline].

3.   Alvarez, LJ, Turner HC, Schutte M, and Candia OA. Localization of a Na-2Cl-K cotransporter in the rabbit lens (Abstract). Invest Ophthalmol Vis Sci 41, Suppl: S863, 2000[ISI].

4.   Anderson, EI, and Fischbarg J. Biphasic effects of insulin and ouabain on fluid transport across rabbit corneal endothelium. J Physiol (Lond) 275: 377-389, 1978[Abstract].

5.   Bildin, VN, Iserovich P, and Fischbarg J. Comparative amounts of membrane transport protein mRNAs in bovine corneal epithelium and endothelium (Abstract). Invest Ophthalmol Vis Sci 37: S1106, 1996.

6.   Bonanno, JA, and Giasson C. Intracellular pH regulation in fresh and cultured bovine corneal endothelium. II. Na+:HCO<SUB>3</SUB><SUP>−</SUP> cotransport and Cl-/HCO<SUB>3</SUB><SUP>−</SUP> exchange. Invest Ophthalmol Vis Sci 33: 3068-3079, 1992[Abstract].

7.   Diecke, FPJ, Wen Q, Kuang K, and Fischbarg J. Regulation of Na+-K+-2Cl- cotransport of cultured bovine corneal endothelial cells (Abstract). Exp Eye Res 67: S127, 1998.

8.   Diecke, FPJ, Zhu Z, Kang F, Kuang K, and Fischbarg J. Sodium, potassium, two chloride cotransport in corneal endothelium: characterization and possible role in volume regulation and fluid transport. Invest Ophthalmol Vis Sci 39: 104-110, 1998[Abstract].

9.   Dikstein, S, and Maurice DM. The metabolic basis of the fluid pump in the cornea. J Physiol (Lond) 221: 29-41, 1972[ISI][Medline].

10.   Fischbarg, J. Active and passive properties of the rabbit corneal endothelium. Exp Eye Res 15: 615-638, 1973[ISI][Medline].

11.   Forbush, B, III, and Palfrey HC. [3H]bumetanide binding to membranes from dog kidney outer medulla. Relationship to the Na,K,2Cl cotransport system. J Biol Chem 258: 11787-11792, 1983[Abstract/Free Full Text].

12.   Giesen-Crouse, EM, and McRoberts JA. Coordinate expression of piretanide receptors and Na+,K+,Cl- cotransport activity in Madin-Darby canine kidney cell mutants. J Biol Chem 262: 17393-17397, 1987[Abstract/Free Full Text].

13.   Haas, M, and Forbush B, III. [3H]bumetanide binding to duck red cells. Correlation with inhibition of (Na+K+2Cl) cotransport. J Biol Chem 261: 8434-8441, 1986[Abstract/Free Full Text].

15.   Haas, M, Johnson LG, and Boucher RC. Regulation of Na-K-Cl cotransport in cultured canine airway epithelia: a [3H]bumetanide binding study. Am J Physiol Cell Physiol 259: C557-C569, 1990[Abstract/Free Full Text].

16.   Hoffmann, EK, Schiodt M, and Dunham P. The number of chloride-cation cotransport sites on Ehrlich ascites cells measured with [3H]bumetanide. Am J Physiol Cell Physiol 250: C688-C693, 1986[Abstract/Free Full Text].

17.   Jelamskii, S, Sun XC, Herse P, and Bonanno JA. Basolateral Na+-K+-2Cl- cotransport in cultured and fresh bovine corneal endothelium. Invest Ophthalmol Vis Sci 41: 488-495, 2000[Abstract/Free Full Text].

18.   Lowry, OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

19.   Lytle, C, Xu JC, Biemesderfer D, and Forbush B, III. Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269: C1496-C1505, 1995[Abstract/Free Full Text].

20.   Maurice, D. The cornea and sclera. In: The Eye, edited by Davson H.. New York: Academic, 1969, p. 489-600.

21.   McLean, IW, and Nakane PK. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22: 1077-1083, 1974[ISI][Medline].

22.   Narula, PM, Xu M, Kuang K, Akiyama R, and Fischbarg J. Fluid transport across cultured bovine corneal endothelial cell monolayers. Am J Physiol Cell Physiol 262: C98-C103, 1992[Abstract/Free Full Text].

23.   O'Donnell, ME. [3H]bumetanide binding in vascular endothelial cells. Quantitation of Na-K-Cl cotransporters. J Biol Chem 264: 20326-20330, 1989[Abstract/Free Full Text].

24.   O'Donnell, ME, and Owen NE. Reduced Na-K-Cl cotransport in vascular smooth muscle cells from spontaneously hypertensive rats. Am J Physiol Cell Physiol 255: C169-C180, 1988[Abstract/Free Full Text].

25.   Raat, NJ, Delpire E, Van Os CH, and Bindels RJ. Culturing induced expression of basolateral Na-K-2Cl cotransporter BSC2 in proximal tubule, aortic endothelium, and vascular smooth muscle. Pflügers Arch 431: 458-460, 1996[ISI][Medline].

26.   Randall, J, Thorne T, and Delpire E. Partial cloning and characterization of Slc12a2: the gene encoding the secretory Na+-K+-2Cl- cotransporter. Am J Physiol Cell Physiol 273: C1267-C1277, 1997[Abstract/Free Full Text].

27.   Riley, MV, Winkler BS, Starnes CA, and Peters MI. Fluid and ion transport in corneal endothelium: insensitivity of modulators of Na+-K+-Cl- cotransport. Am J Physiol Cell Physiol 273: C1480-C1486, 1997[Abstract/Free Full Text].

28.   Tao, W, Wu X, Liou GI, Abney TO, and Reinach PS. Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells. Invest Ophthalmol Vis Sci 38: 130-141, 1997[Abstract].

29.   Trenberth, SM, and Mishima S. The effect of ouabain on the rabbit corneal endothelium. Invest Ophthalmol Vis Sci 7: 44-52, 1968.

30.   Turner, RJ, and George JN. Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site. J Membr Biol 113: 203-210, 1990[ISI][Medline].

31.   Usui, T, Seki G, Amano S, Oshika T, Miyata K, Kunimi M, Taniguchi S, Uwatoko S, Fujita T, and Araie M. Functional and molecular evidence for Na+-HCO<SUB>3</SUB><SUP>−</SUP> cotransporter in human corneal endothelial cells. Pflügers Arch 438: 458-462, 1999[ISI][Medline].

32.   Yerby, TR, Vibat CR, Sun D, Payne JA, and O'Donnell ME. Molecular characterization of the Na-K-Cl cotransporter of bovine aortic endothelial cells. Am J Physiol Cell Physiol 273: C188-C197, 1997[Abstract/Free Full Text].

33.   Zhu, Z, Kuang K, Kang F, Li J, and Fischbarg J. Platelet activating factor inhibits fluid transport by corneal endothelium. Invest Ophthalmol Vis Sci 37: 1899-1906, 1996[Abstract].


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