Eye Research Institute, Oakland University, Rochester, Michigan 48309-4401
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ABSTRACT |
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The role of
Na+-K+-2Cl
cotransport in ion and fluid transport of the corneal endothelium was
examined by measuring changes in corneal hydration and uptake of
86Rb by the endothelial cell
layer. Isolated, intact rabbit corneas maintain normal hydration when
they are superfused at the endothelial surface with bicarbonate
(
)-Ringer solutions as a
result of equilibrium between active ion and fluid transport out of the
stromal tissue and leak of fluid into stromal tissue from the aqueous
humor. Furosemide and bumetanide did not alter this equilibrium when
they were added to the superfusion medium. Uptake of
86Rb by the endothelium of the
incubated cornea was increased 25% by bumetanide, but uptake in the
presence of ouabain (70% less than that of controls) was not changed
by bumetanide. In Na+-free medium,
uptake of 86Rb was reduced by
58%, but it was unchanged in
Cl
-free medium. Calyculin
A, a protein phosphatase inhibitor and activator of
Na+-K+-Cl
cotransport, was without effect on
86Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes was
significantly different when bumetanide was present in the media. It is
concluded that
Na+-K+-2Cl
cotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport of
this cell layer. Its presence in cultured endothelial cells may reflect
the reported importance of this protein in growth, proliferation, and
differentiation.
sodium; potassium; chloride; bumetanide; rubidium-86 uptake; corneal hydration; calyculin A; hypertonicity
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INTRODUCTION |
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IT IS WELL ESTABLISHED that removal of
Na+ from the medium bathing the
endothelial cells of the cornea causes failure of the ion transport
processes that regulate corneal hydration, resulting in pronounced
swelling of the tissue (7, 10). Because identical results were obtained
in the presence of ouabain, this effect has been attributed to the
requirement for Na+ in the
function of
Na+-K+-adenosinetriphosphatase
(ATPase), the enzyme that couples energy supply and active transport
(22, 29). It has also been shown that both bicarbonate
() and
Cl
are critical to normal
function of the fluid-transporting capacity of these cells (10, 32).
Several models of interacting ion fluxes have been proposed, and
experimental evidence has been obtained that indicates that
Na+/H+
and
Cl
/
exchange channels,
Na+-
cotransport channels, and
Cl
channels may be
important components in the regulation of water movement across these
cells (3, 8, 12). More recently, the involvement of
Na+-K+-2Cl
cotransport has also been proposed on the basis of the observation of
bumetanide-sensitive 86Rb uptake
in cultured endothelial cells (6). However, furosemide, a loop diuretic
acting similarly to bumetanide (9, 17), was shown in an earlier study
(12) to have no effect on the intracellular potential of cultured
cells, and both of these compounds have been shown not to affect the
hydration of superfused corneas (32), suggesting
Na+-K+-2Cl
cotransport is not a critical component of transendothelial fluid movement.
In view of this conflicting evidence regarding a possible role for the
Na+-K+-2Cl
cotransporter in the corneal endothelium, we examined both
physiological and biochemical responses of the intact, isolated cornea
when it is exposed to the loop diuretics and to other conditions, such as anisotonicity, phosphatase inhibition, and adenosine
3',5'-cyclic monophosphate (cAMP) activation, that are
known to modulate
Na+-K+-2Cl
transport in other tissues (4, 11, 14). The measurement of corneal
thickness is an assessment of the fluid transport capacity of the
endothelium, and the uptake of
86Rb is a measure of the ability
of cells to accumulate K+ against
a concentration gradient, a process requiring metabolic energy or other
existing ion gradients.
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MATERIALS AND METHODS |
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Cornea preparation. New Zealand White rabbits weighing 1.8-2.5 kg were maintained on lab chow and water ad libitum and were killed by intravenous injection of Euthanasia-5 solution (Veterinary Laboratories, Lenexa, KS), and their eyes were enucleated with the lids. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1985). Corneas were then isolated and mounted for superfusion of the endothelial surface and measurement of thickness with the specular microscope by the method of Dikstein and Maurice (7), using silicone oil (200 fluid, Dow Corning, Midland, MI) on the anterior, tear-side surface. The thickness of the cornea has been shown to be directly proportional to its hydration because it is inelastic in the tangential plane; consequently, when oil covers the anterior surface, monitoring of thickness provides a measure of net water flux across the posterior, endothelial surface (16). The initial readings of thickness were made within 3 min of the start of the superfusion (flow rate, 3 ml/h; temperature, 35°C), and subsequent readings were made every 20 min for periods up to 5 h. Initial values, ranging from 380 to 408 µm, were normalized to 400 µm in Figs. 1 and 2.
Superfusion.
The control medium was a Krebs-Ringer
solution containing (in mM) 122 NaCl, 25 NaHCO3, 6.7 KCl, 1.0 CaSO4, 0.8 Na2
HPO4, 0.6 MgSO4, and 5.5 glucose and 40 mg/l
gentamicin. The pH was 7.4 after the solution was bubbled with 5%
CO2-7%
O2-88% N2, and the osmolality was
284-290 mosmol/kgH2O
(Advanced Instruments, Needham Heights, MA). Experiments were also done
in 43 mM
-Ringer solution,
substituting equimolar NaHCO3 for
NaCl, with a resulting pH of 7.7 after equilibration with 5%
CO2. Changes in tonicity of this
medium were made by omitting 32 mM NaCl (hypotonic, 226 ± 3 mosmol/kgH2O) and by adding 32 mM
NaCl or 60 mM sucrose (hypertonic, 345 ± 3 mosmol/kgH2O). Solution changes at
the endothelial surface were made by exchanging syringes on the Sage
pumps (Orion Research, Cambridge, MA) and by flushing the chamber with
1 ml of the new solution, about four turnovers of chamber volume.
86Rb uptake.
Corneas were cut from the eyeball at the limbus and were dipped in
-Ringer solution to remove aqueous humor. They were then preincubated for 10 min in appropriate nonlabeled media before being transferred to vials containing 3.6 ml media with
[3H]mannitol (19.7 Ci/mmol),
[14C]urea (55 mCi/mmol), and
86Rb-labeled rubidium chloride
(1.1 mCi/mg), all obtained from NEN DuPont (Boston, MA), at
concentrations of 2.5, 1, and 0.5 µCi/ml, respectively, for periods
of 7-60 min. The control media were the same 25 mM and 43 mM
-Ringer solutions used for
superfusion. For Cl
-free or
-free media,
Cl
was replaced in the 25 mM
-Ringer solutions by gluconate
or nitrate, and
was replaced by
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, maintaining K+, pH, and
osmolality constant. Na+-free
medium was prepared by substitution of tetramethylammonium chloride for
NaCl and choline bicarbonate for
NaHCO3. Hyperosmotic and
hyposmotic media were obtained by the same procedures described in
Superfusion above.
Determination of 86Rb uptake. [3H]mannitol was used as a marker for the distribution of the isotopes in the extracellular space (intercellular plus surface residue) after rinsing, the tissue-to-medium ratio (T/M) of 3H disintegrations per minute (dpm) in the endothelial sample to 3H dpm in 1 µl of incubation medium being the "apparent extracellular volume" (AEV). Thus AEV × dpm of 1 µl 86Rb incubation medium is the extracellular 86Rb in the sample scraped from the cornea. Subtracting this value from total 86Rb in the sample gives the dpm accumulated within the cell. The uptake of K+ is calculated (in nmol) from the specific activity of the 86Rb and K+ concentration of 6.7 mM. Intracellular volume is derived by subtracting the 3H T/M value (AEV) from the 14C T/M value, which is a measure of total fluid in the sample, urea being freely permeable. Thus the intracellular K+ concentration and T/M are obtained. [Note: the T/M values for 7-min incubations shown in Fig. 3 have been adjusted downward by 32% to allow for an artificially low calculated intracellular volume (0.33 ± 0.17 µl at 7 min vs. 0.49 ± 0.15 µl at 30 and 60 min; P < 0.05) that resulted from an incomplete penetration of [14C]urea into the intracellular compartment over 7 min.] Values are means ± SD, and P values were obtained with Student's two-tailed t-test.
Cultured cells.
86Rb uptake was also measured in
cultured retinal pigment epithelial (RPE) cells. Primary cultures of
human RPE cells were passaged four to five times and were plated in
12-well plates. At confluence, the medium (Dulbecco's modified
Eagle's medium with 10% calf serum, GIBCO, Gaithersburg, MD) was
aspirated and replaced with 1 ml of 25 mM
-Ringer solution (see
Superfusion above) for 60 min. This medium was aspirated and replaced for a 10-min preincubation
period with the same medium containing appropriate additions. This was
replaced with identical medium containing
[3H]mannitol and
86Rb (4 and 1 µCi/ml,
respectively) for a 30-min incubation. Medium was then decanted, and
the plate was immersed and gently agitated in 1 liter of ice-cold
K+-free medium. After the plate
was drained, 0.5 ml 0.1 N NaOH was added to the wells for 30 min with
shaking. Samples of 0.2 and 0.02 ml were taken from each well for
counting and protein assay, respectively. Values of
86Rb dpm were corrected for
extracellular (residual) fluid in the wells in proportion to the
residual [3H]mannitol
dpm values. The correction was equivalent to ~0.16 µl fluid and was
<10% of the dpm recovered in the wells with the smallest total
86Rb uptake (i.e., with ouabain
plus bumetanide).
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RESULTS |
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Superfusion.
In the control 25 mM superfusion
medium, corneas behaved in the manner described previously (24), swelling over the first hour to reach a new steady-state thickness ~25 µm above the initial value (Fig.
1A). Bumetanide (up to 1 mM) and
furosemide at 100 µM did not alter this pattern, whereas furosemide
at 1 mM caused corneas to swell slowly beyond the new equilibrium
thickness seen in controls. In 43 mM
-Ringer solution, corneas
maintained their initial thickness for >3 h (Fig.
1B). Again, bumetanide, at
concentrations from 1 µM to 1 mM had no effect on corneal thickness,
and, at this more optimal
concentration (23), furosemide was also without effect from 1 µM to 1 mM.
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86Rb uptake.
In preliminary experiments, with use of the triple-label method, it was
established that 86Rb was
accumulated by the endothelial cells of the incubated corneas. The rate
was not linear, but accumulation increased for >1 h and was markedly
inhibited by the presence of
103 M ouabain (Fig.
3), and, in a competitive manner, by
varying K+ concentrations.
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86Rb uptake by cultured cells.
In consideration of the failure of bumetanide to inhibit
86Rb uptake by the endothelial
cells of the cornea in these circumstances, we measured the effect of
bumetanide on an available cultured cell model that has been shown to
exhibit bumetanide-sensitive 86Rb
uptake (13). The data for 86Rb
uptake by cultured RPE cells are shown in Fig.
6. Bumetanide at 100 µM inhibited uptake
by 50% and, together with ouabain, eliminated virtually all
86Rb accumulation. Exposure of the
cells to hypertonic conditions increased uptake by 20%, and this
increase was also eliminated by bumetanide. Uptake in the control
condition was 8.6 ± 1.07 nmol · mg
protein1 · min
1.
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DISCUSSION |
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The results shown in Fig. 1 confirm and extend previously obtained data
that indicate that the loop diuretics bumetanide and furosemide have no
detectable effect on the control of hydration of isolated, superfused
corneas (32). Only at the relatively high concentration of 1 mM did
furosemide, the less specific of the two compounds (17), cause a small
degree of swelling in corneas superfused with 25 mM
-Ringer solution. These results
suggest that the
Na+-K+-2Cl
cotransporter does not play a role in the regulation of fluid movement
across the rabbit corneal endothelium under normal circumstances. This
conclusion is surprising in consideration of the demonstrations of a
ouabain-insensitive, bumetanide-sensitive
K+ uptake in cultured bovine
corneal endothelial cells that is proposed to play a role in cellular
volume regulation and hence, perhaps, in vectorial fluid transport (6,
25, 33). It is, however, supported by a recent finding that plasma
membrane vesicles prepared from bovine corneal endothelium showed no
evidence of Na+ transport coupled
to K+ and
Cl
(15). It is possible
that the cotransporter operates in cellular ion homeostasis (without
any short-term impact on corneal thickness) rather than being an
integral component of transendothelial fluid flux. Alternatively,
differences in its expression may reflect a species difference (rabbit
vs. bovine) or a difference between the use of fresh tissue versus
cultured cells. Indeed, several reports indicate that the activity of
this cotransporter specifically is present in cultured cells but
missing in their respective native tissues (2, 30) and is present in
growing cells but not differentiated ones (5, 18). Similarly, cells of
fetal RPE tissue exhibit specific
K+ currents that are absent in the
RPE of adult tissue (31), again indicating loss of a membrane transport
protein during development.
We therefore attempted to demonstrate a
K+ uptake process directly in the
native endothelium and determine if it had the same characteristics as
that found in cultured bovine cells. Figure 4 shows that the
intracellular concentration of
86Rb was 4.8-fold greater than
that of the incubation medium after 30 min, or 32.2 mM. In the presence
of ouabain, the accumulation was reduced to 1.5-fold, an inhibition of
70%. Furosemide and bumetanide each increased
86Rb uptake, the latter by a
significant 25% (P < 0.01), but
neither compound altered the uptake found in the presence of ouabain. These data indicate the absence of ouabain-insensitive,
bumetanide-sensitive K+ uptake, a
definitive characteristic of the cotransporter. These results are
clearly different from those seen in the cultured human RPE cells, in
which bumetanide inhibited 86Rb
uptake by 50% and had an additive inhibitory effect to that of
ouabain. This response is essentially the same as that reported by
Diecke et al. (6) in cultured bovine corneal endothelial cells and is
typical of 86Rb uptake patterns in
other ocular cells, such as frog and human RPE (1, 13) and human
nonpigmented ciliary epithelium (4), that have been ascribed to the
Na+-K+-2Cl
cotransporter.
The cotransporter, as its name implies, is further defined by the
requirement for the simultaneous presence of all three ions to
function. Thus the ability of the corneal endothelium to maintain control levels of 86Rb uptake in
the absence of Cl, whether
replaced by gluconate or by nitrate, is further strong evidence arguing
for the absence of a functional cotransporter in these cells. Moreover,
the removal of Na+ inhibited
uptake to virtually the same extent as ouabain, suggesting that
K+ accumulation in this cell layer
is almost entirely via the
Na+-K+-ATPase.
The activity of the cotransporter is increased by phosphorylation, and
86Rb uptake is thus found to be
stimulated by inhibitors of protein phosphatases such as calyculin A
(14), but this agent was without effect on uptake by the endothelium.
Because the activity of the
Na+-K+-2Cl
cotransporter has been linked to compensatory changes in cell volume
after exposure to hypertonic and hypotonic media (11, 14), experiments
were carried out to evaluate the changes in stromal thickness and
86Rb uptake in response to changes
in osmolality. As shown in Fig. 2, hypertonic superfusion conditions
caused decreases of corneal thickness that were followed by a trend to
revert to initial thickness. The initial changes in stromal thickness
most likely result from paracellular movement of fluid in response to
the imposed osmotic gradients, in a manner analogous to the change in
corneal thickness seen when
Cl
is replaced by an
impermeant ion such as gluconate. That response also was ascribed to an
osmotic effect, in that case, as a result of differences in the
reflection coefficients of the two anions (32). In the
hypertonic-treated cornea, the osmotic pressure of the medium is higher
than that of the stroma, and fluid moves from the stroma to medium,
thinning the cornea. In the hypotonic-treated cornea, the osmotic
gradient is reversed, and water moves from medium to stroma, causing
the cornea to swell. These changes in hydration of the cornea in
response to anisotonic loads are consistent with the fact that fluid
balance is dependent on the Na+
activity gradient between aqueous humor and stroma (28).
The subsequent corrective (regulatory) changes in thickness toward the
initial value must depend on changes in cellular ion and fluid transport, permeability of the cell layer, or swelling pressure of the
stromal matrix. Although all are possible, the first two would result
from volume regulation in the endothelium and the last would result
from changes in the activities of ions in the stroma (28), and it is
not yet clear which might be the major contributor to reversal of the
initial response to anisotonic media. Interestingly, these patterns of
recovering original thickness in the face of continued exposure to the
osmotically altered media are reminiscent of the cellular volume
changes seen under conditions known as regulatory volume increase (RVI)
and decrease (RVD) (27). These terms are applied to the corneal events
only in a descriptive sense at this time, in recognition that the
analogy to cellular volume regulation is neither proven nor complete.
Corneas responded to hypotonic conditions with only a slow and
incomplete recovery in control medium, but in the presence of forskolin
or adenosine the rate of thinning was increased and the cornea
equilibrated close to its initial thickness. These compounds have been
shown to increase net fluid transport across the corneal endothelium by
activation of adenosine A2B
receptors and stimulation of adenylylcyclase, leading to an increase of cAMP in the endothelial cells (24). Bonanno and Srinivas (3, 27) have
found that cAMP stimulated
Cl transport of bovine
corneal endothelial cells and that
Cl
channels were activated
by hypotonically-induced swelling, which suggests that the RVD-type
response of the cornea in the presence of adenosine might depend on
these anion channels. The comparable thinning of the cornea in response
to bumetanide could also depend on channel activation, because it has
been shown in vascular endothelial cells that RVD, which results from
efflux of ions via KCl cotransport and
K+ channels, is also stimulated by
bumetanide (21).
The RVI of cells exposed to hypertonic conditions is believed to result
from stimulation either of
Na+-K+-2Cl
cotransporters or of coupled
Na+/H+
and
Cl
/
exchangers that increase the ionic content of the cells, resulting in
water influx to restore volume (11). The former process typically is
inhibited in the presence of bumetanide (11). However, in the cornea,
the increase in thickness subsequent to hypertonic thinning was
unaltered by bumetanide. Moreover, although the uptake of
86Rb in hypertonic medium was
increased by only 12%, (P = 0.1), this increase in uptake was not inhibited by bumetanide but was, in
fact, increased by a further 18% (P < 0.02). In the cultured RPE cells, on the other hand, a different
pattern of 86Rb uptake was seen on
exposure to hypertonic media. The increase of 20% in media of 345 mosmol/kgH2O was entirely
eliminated by bumetanide, confirming that the response to hypertonicity
was dependent on increased
Na+-K+-2Cl
cotransporter activity. In cultured corneal endothelial cells, a 35%
increase of bumetanide-sensitive
86Rb uptake was reported in
hypertonic conditions, and, in hypotonic conditions, there was a 40%
decrease (6). In the native endothelium, it can be seen that only
hypotonicity caused a significant change in
86Rb uptake, but this was little
altered by bumetanide.
In summary, the lack of effect of bumetanide on the equilibrium
thickness of corneas in -Ringer
solution and the insensitivity of
86Rb uptake by the endothelium to
bumetanide and calyculin A and to the removal of
Cl
indicate that operation
of an
Na+-K+-2Cl
cotransporter is not integral to the normal function of the corneal endothelium. This conclusion distinguishes the characteristics of the
ion-transport mechanisms of the intact endothelium of the in vitro
rabbit cornea from those of bovine cultured endothelial cells. It
might be speculated that the endothelium, being a terminally differentiated and virtually nondividing cell population, has lost the
ability to express the
Na+-K+-2Cl
cotransporter, a membrane protein whose activity appears to be particularly important in growth, differentiation, and proliferation, processes active in the preparation of cell cultures (5, 19, 20).
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ACKNOWLEDGEMENTS |
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The authors thank Diane Wilson and Aaron Heilbrun for preparation of the cultured RPE cells, Alice M. Carleton for preparation of the manuscript, and Dr. Jason R. Lane for permission to cite work in press.
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FOOTNOTES |
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This work was supported in part by National Eye Institute Grants EY-00541 and EY-10015 and by the Core Grant for Vision Research, EY-05230.
Address reprint requests to M. V. Riley.
Received 2 May 1997; accepted in final form 7 July 1997.
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