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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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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
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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 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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RESULTS |
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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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[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 × 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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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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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 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
buffer was substituted for HEPES-HCO
(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
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.
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DISCUSSION |
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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 protein1 · 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
KK + K
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
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ACKNOWLEDGEMENTS |
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We acknowledge technical advice from Dr. C. Lytle.
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FOOTNOTES |
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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.
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