Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na+-K+-2Clminus cotransport

Michael V. Riley, Barry S. Winkler, Catherine A. Starnes, and Margaret I. Peters

Eye Research Institute, Oakland University, Rochester, Michigan 48309-4401

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (HCO<SUP>−</SUP><SUB>3</SUB>)-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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (HCO<SUP>−</SUP><SUB>3</SUB>) 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-/HCO<SUP>−</SUP><SUB>3</SUB> exchange channels, Na+-HCO<SUP>−</SUP><SUB>3</SUB> 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 HCO<SUP>−</SUP><SUB>3</SUB> 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 HCO<SUP>−</SUP><SUB>3</SUB>-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 HCO<SUP>−</SUP><SUB>3</SUB>-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 HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solutions used for superfusion. For Cl--free or HCO<SUP>−</SUP><SUB>3</SUB>-free media, Cl- was replaced in the 25 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solutions by gluconate or nitrate, and HCO<SUP>−</SUP><SUB>3</SUB> 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.

We terminated incubations by rinsing the cornea in ice-cold incubation medium lacking isotopes and all K+ to limit any K+/86Rb+ exchange (6 dips, followed by draining, repeated in 2 separate 12-ml volumes). Endothelium (without Descemet's membrane) was then scraped off with a scalpel blade, transferred to 5.0 ml scintillation fluid (Beckman Ready-Safe, Arlington Heights, IL), vortexed, and sonicated. Differential counting of 3H, 14C, and 86Rb was done in a Beckman LS 5801.

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 HCO<SUP>−</SUP><SUB>3</SUB>-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).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Superfusion. In the control 25 mM HCO<SUP>−</SUP><SUB>3</SUB> 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 HCO<SUP>−</SUP><SUB>3</SUB>-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 HCO<SUP>−</SUP><SUB>3</SUB> concentration (23), furosemide was also without effect from 1 µM to 1 mM.


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Fig. 1.   Changes in corneal thickness during superfusion in HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution in presence or absence of loop diuretics; n = 3. A: 25 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution. +, Control (Ctl) medium with no additions; square , with 100 µM bumetanide; open circle , with 1 mM furosemide. B: 43 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution. +, Ctl medium with no additions; open circle , with 1 mM furosemide. Values are means ± SD; initial readings of all corneas were normalized to 400 µm.

The effects of changing the osmolality of the superfusing solutions are shown in Fig. 2. These experiments were all done in 43 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution so that changes in corneal thickness resulting from changes in osmolality were not superimposed on or confused by the basal swelling seen in control 25 mM HCO<SUP>−</SUP><SUB>3</SUB> media. On exposure of the endothelium to the 43 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution of 345 mosmol/kgH2O, the cornea thinned rapidly by 50-60 µm and then began to swell, approaching its original thickness at a rate of 20-25 µm/h (Fig. 2A). The cornea did not fully equilibrate at its original thickness, and the swelling rate merely slowed or halted temporarily at this point before swelling continued. Bumetanide (100 µM) did not alter any phase of this pattern of thinning and swelling.


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Fig. 2.   Changes in corneal thickness on changing osmolality of 43 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution in presence or absence of loop diuretics or with forskolin (n = 3). A: hypertonic medium (345 mosmol/kgH2O) introduced at arrow. B and C: hypotonic medium (226 mosmol/kgH2O) introduced at arrow. Values are same as for Fig. 1.

Under hypotonic conditions, the cornea swelled rapidly by 50-60 µm and then deturgesced very slowly over the next 3 h (Fig. 2B). In contrast, when 100 µM bumetanide was present in the hypotonic medium, after swelling 50-60 µm, corneas immediately began to deturgesce until the original thickness was reached or approached and then maintained. This effect of bumetanide was dose dependent, with lower concentrations causing corneas to reach equilibrium thicknesses only part way toward complete deturgescence; at 10 µM, recovery was ~50% of that at 100 µM and 20% of that at 1 µM (data not shown). Addition of 50 µM forskolin (or 100 µM adenosine, not shown), promoters of net endothelial fluid transport (24), also increased the rate and extent of deturgescence in a hypotonic medium in essentially the same manner as bumetanide (Fig. 2C). Furosemide at 100 µM increased deturgescence to a small extent, comparable to 1 µM bumetanide.

Calyculin A, an inhibitor of protein phosphatases, was added to superfusion medium to assess its effect on fluid movement across the endothelium. In isosmotic medium at 10 nM, after a lag period of ~45 min, calyculin A caused swelling at 25-35 µm/h (n = 3). At 100 nM, swelling was immediate and rapid (100-200 µm/h) and the typical specular image of the endothelial cells was destroyed. Okadaic acid, another protein phosphatase inhibitor, had similar effects but at concentrations 3- to 10-fold higher than calyculin A.

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 10-3 M ouabain (Fig. 3), and, in a competitive manner, by varying K+ concentrations.


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Fig. 3.   Time course of changes in tissue-to-medium ratio (T/M) of 86Rb in endothelium of intact corneas after incubation in 25 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution containing 86RbCl, [14C]urea, [3H]mannitol, and different concentrations of K+: A, 1 mM; B, 2 mM; C, 6.7 mM (Ctl); D, 6.7 mM + 1 mM ouabain. Values are means ± SD; n = 3 for A, B, and D, and n = 6 for C.

The effects of bumetanide, furosemide, and calyculin A and of the elimination of Cl- or Na+ on 86Rb uptake over 30 min are shown in Fig. 4, together with the effect of ouabain in these conditions. At 100 µM, the loop diuretics each increased the 86Rb uptake, furosemide by 13% (nonsignificant) and bumetanide by 25% (P < 0.01), but, in the simultaneous presence of ouabain, the uptake was not different from that found in ouabain-treated controls. Neither calyculin A at 100 nM nor substitution of Cl- with gluconate or NO<SUP>−</SUP><SUB>3</SUB> altered 86Rb uptake. When Na+ in the medium was replaced by tetramethylammonium chloride and choline ions, 86Rb was reduced from the control T/M value of 4.8 to 2.2 (P < 0.01). Addition of ouabain to the Na+-free medium reduced the T/M value slightly further to 1.7, consistent with all other ouabain data.


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Fig. 4.   Effect of different conditions on T/M of 86Rb in endothelium after 30-min incubations in 25 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution in the absence (open bars) and presence (hatched bars) of 1 mM ouabain. Ctl, 25 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution with 6.7 mM K+ and no additions; Frs, Ctl + 100 µM furosemide; Bmt, Ctl + 100 µM bumetanide; Cal A, Ctl + 100 µM calyculin A; 0 Cl-, Ctl with chloride replaced by NO<SUP>−</SUP><SUB>3</SUB> or by gluconate (Gl-); 0 Na+, Ctl with sodium replaced by tetramethylammonium and choline. Values are means ± SD; n = 4-10. * Significantly different from Ctl at P < 0.05; **significantly different from Ctl at P < 0.01.

Increasing the osmolality of the incubation medium by 60 mosmol/kgH2O increased 86Rb uptake slightly, although not statistically significantly, whereas decreasing osmolality to 226 mosmol/kgH2O reduced uptake by 35% (P < 0.01) (Fig. 5). Under these anisotonic conditions, the simultaneous presence of ouabain resulted in the same inhibition of uptake as in all other conditions (not shown). No difference was observed in these results, whether hypertonic conditions were achieved by adding 32 mM NaCl or 60 mM sucrose. Note that in Fig. 5 all experiments were carried out in 43 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution to parallel the conditions used for the anisotonic superfusion experiments; uptake in 43 mM HCO<SUP>−</SUP><SUB>3</SUB> was slightly greater than that in 25 mM HCO<SUP>−</SUP><SUB>3</SUB> (Fig. 4). Bumetanide at 100 µM increased the uptake of 86Rb under both the hypertonic and hypotonic conditions. In medium of 346 mosmol/kg with bumetanide, uptake was significantly greater than in the 43 mM HCO<SUP>−</SUP><SUB>3</SUB> controls (P < 0.05).


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Fig. 5.   T/M of 86Rb in endothelium of corneas incubated in anisotonic medium containing 43 mM HCO<SUP>−</SUP><SUB>3</SUB> with or without 100 µM Bmt. Ctl, 285 mosmol/kgH2O; Hyper, 345 mosmol/kgH2O with 60 mM sucrose; Hypo, 226 mosmol/kgH2O with depleted NaCl; n = 8 for Ctl, Hyper, and Hyper + Bmt, and n = 4 for Hypo and Hypo + Bmt. Values are means ± SD. * Significantly different from control at P < 0.05; ** significantly different from control at P < 0.01.

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 protein-1 · min-1.


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Fig. 6.   86Rb uptake by cultured retinal pigment epithelial (RPE) cells incubated for 30 min in 25 mM HCO<SUP>−</SUP><SUB>3</SUB>-Ringer solution. Ctl, no additions; Ou, with 1 mM ouabain; Bmt, with 100 µM bumetanide; Ou + Bmt, with 1 mM ouabain + 100 µM bumetanide; Hyper, in hypertonic medium (345 mosmol/kgH2O); Hyper + Bmt, in hypertonic medium with 100 µM bumetanide. Values are means ± SD; n = 4. * Significantly different from control at P < 0.05; ** significantly different from control at P < 0.01; # significantly different from Ou or Bmt at P < 0.01; + significantly different from Hyper at P < 0.01.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 HCO<SUP>−</SUP><SUB>3</SUB>-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-/HCO<SUP>−</SUP><SUB>3</SUB> 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 HCO<SUP>−</SUP><SUB>3</SUB>-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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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