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Am J Physiol Cell Physiol 290: C1168-C1178, 2006; doi:10.1152/ajpcell.00254.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Regulation of water permeability in rabbit conjunctival epithelium by anisotonic conditions

Oscar A. Candia,1,2 Lawrence J. Alvarez,1 and Aldo C. Zamudio1

1Department of Ophthalmology and 2Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York

Submitted 26 May 2005 ; accepted in final form 22 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of unilateral exposure to anisotonic conditions on diffusional water permeability of the isolated rabbit conjunctiva were determined. A segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes (Jdw) were measured in either direction by adding 3H2O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. Jdw were determined under control isosmotic conditions and after introduction of either hyper- or hypotonic solutions against the tear or stromal sides of the preparations. In each of these four separate conditions, the anisotonic medium produced an ~20–30% reduction in Jdw across the tissue, with the exception that to obtain such reduction with increased tonicity from the stromal side (medium osmolality increased by adding sucrose), conditions presumptively inhibiting regulatory volume increase mechanisms (e.g., pretreatment with amiloride and bumetanide) were also required. All reductions in Jdw elicited by the various anisotonic conditions were reversible on restoration of control tonicity. In experiments in which preparations were pretreated with the protein cross-linking agent glutaraldehyde, anisotonicity-elicited reductions in Jdw were not observed. Such reductions were also not observed in the presence of HgCl2, implying the involvement of aquaporins. However, it is possible that the mercurial may be toxic to the epithelium, preventing the tonicity response. Nevertheless, from concomitant changes in transepithelial electrical resistance, as well as [14C]mannitol fluxes, [14C]butanol fluxes, and Arrhenius plots, arguments are presented that the above effects are best explained as a cell-regulated reduction in membrane water permeability that occurs at the level of water-transporting channels. Presumably both apical and basolateral membranes can downregulate their water permeabilities as part of a protective mechanism to help maintain cell volume.

unidirectional water fluxes; net water fluxes; Ussing chamber; short-circuit current; electrolyte transport; cell volume regulation; paracellular mannitol permeability

A MAJOR ADVANCE IN THE STUDY of fluid transport came from the discovery of water channels or pore proteins now known as aquaporins (AQPs). Extensive work has identified, sequenced, and localized the individual water channels in an attempt to determine their function (19). Other membrane-spanning proteins such as CFTR, some classes of K+ channels, and the glucose transporter exist in sufficient abundance that they might also contribute to transcellular water movement (22, 25). In addition, the lipid bilayer of the cell membrane allows a substantial flow of water, but unlike protein channels the bilayer is less susceptible to solute interaction and regulation.

We viewed the impressive structural, chemical, and molecular details of water channels that have come to light as insightful to physiologists for reinterpreting some of the phenomenological data of water fluxes that had remained unexplained. Initially, we (2) reexamined the apparent "rectification" phenomenon that existed in measurements of transepithelial water movement across the amphibian bladder. Briefly, AVP elicits as much as a 40-fold increase in net water fluxes across the bladder when mucosal side osmolarity is diluted 10-fold. However, an AVP induction of a net water flux is not obtained when the osmotic gradient is in the opposite direction (serosal side hypotonic), implying a rectification. However, this condition produces a reduction of unidirectional water fluxes (Jdw) in both directions, thereby indicating a simple decrease in permeability induced by the hypotonic milieu against the serosal aspect, which is observed as a rectification when the osmotic gradient necessary to elicit a net water flux (Jv) is imposed in that particular direction. We posited (2) that the water permeability of the basolateral membrane could have been downregulated when these cells began to swell from their serosal sides, because of either AQP closure or the removal of constitutive water channels from the membrane.

More recently, we examined (3, 5) the water permeability characteristics of the epithelia of frog cornea and rabbit conjunctiva isolated in Ussing-type chambers. Although the corneal epithelium is similar to the bladder in that its apical aspect has a relatively lower water permeability than its basolateral surface (3), such sidedness is not a characteristic of the conjunctiva in that neither the apical nor basolateral surfaces are rate limiting, from comparisons of one face against the other, to transepithelial water movement (5). However, more importantly, it was found that a reduction in Jdw in response to basolateral hypotony was a trait common to these ocular tissues (3, 5), as we initially observed with the amphibian bladder (2). Furthermore, Jdw across the frog corneal epithelium are also inhibited by hypertonic conditions (3), an experimental perturbation that had heretofore remained unexamined with the mammalian epithelium of the rabbit conjunctiva. Therefore, additional experiments are presented here that are based on measurements of Jdw across isolated, short-circuited conjunctival preparations to extend the characterization of this epithelium.

We have argued previously (5) that labeled water will cross cell membranes via all available pathways (lipid bilayer, AQPs, and other channels) and that the flow can be accelerated, or reduced, by affecting any of these pathways. Therefore, osmotic or hydrostatic forces that affect Jv could also affect unidirectional fluxes.

Because Jdw measurements in either direction represent fluxes across the same membrane pathways, they change equally when the water permeability of an epithelium is affected by an experimental maneuver, such as the introduction of unilateral anisotonic conditions. Clearly, the measurement of Jv requires a driving force, either osmotic or hydrostatic. Under these conditions the osmotic permeability coefficient (Pf) is calculated, and it is assumed that it remains the same in the absence of the driving force. However, Pf may change under the influence of different gradients. This incongruity is avoided by measuring Jdw with and without osmotic gradients. When an osmotic perturbation effected on only one side of the membrane affects both unidirectional fluxes, it is an indication that the overall membrane permeability has changed.

Using the conjunctiva as a model system, in the present study we demonstrate the responses of the epithelium to osmolality changes in either bathing solution and present evidence consistent with the likelihood that epithelia have mechanisms (presumably at the level of the AQPs or other water-transporting channels) to regulate their water permeability when confronted with abnormal tonicities. Moreover, indirect evidence was obtained suggesting that membrane water permeability may be downregulated secondarily to changes in cell volume.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The protocols for procuring, isolating, and mounting within modified Ussing-type chambers the epithelium of rabbit conjunctiva were described in detail previously (28). Briefly, adult albino rabbits of either sex weighing 2–3 kg were rapidly killed by CO2 asphyxiation, a protocol approved by the Mount Sinai Animal Care and Use Committee. Treatment of the rabbits before death conformed to the "Guiding Principles in the Care and Use of Animals" of the American Physiological Society. The bulbar-palpebral conjunctiva was dissected as a cylinder and cut longitudinally to convert it to a flat epithelium that was mounted as a partition between the hemichambers, exposing 0.32 cm2 of cross-sectional area. The chambers had the necessary accessories for determinations of transepithelial electrical parameters and vigorous stirring, the latter provided by a continuous bubbling within each hemichamber with a humidified CO2-air gas mixture. Such bubbling should minimize the influence of unstirred layers during the flux experiments described below. Because unstirred layers are in series with the main membrane and assumed to be constant, only the absolute values in calculated fluxes might be affected, not the observed changes elicited by the experimental maneuvers.

Water fluxes. To measure transepithelial Jdw (expressed in text as µl·min–1·cm–2), the approach was identical to that used earlier with the amphibian bladder and cornea (2, 3), as well as in previous work with the rabbit conjunctiva (5). For this, tritiated water was unilaterally added to one compartment (~1 µCi/ml bathing solution). Thereafter, 2-ml samples from the unlabeled, opposite-side bath were drawn every 15 min and replaced with fresh bathing solution, thereby maintaining the chamber volume and preventing the "cold-side" activity from exceeding 5% of that of the radiolabeled side. At least four such cold-side samples were obtained for each experimental condition, with a complete experiment usually consisting of three conditions so that paired comparisons could be made. The labeled side was sampled (25 µl) periodically (usually at 30-min intervals) to determine the specific activity and ensure its constancy. To prevent a significant reduction of the specific activity from the labeled-side bath during experiments of 3- to 4-h duration, the volume of the labeled bath was twice that of the cold side. Samples were mixed with an appropriate fluor and counted in a liquid scintillation detector.

The transconjunctival potential difference was short-circuited (26) during the sampling of the 3H2O fluxes, with the current needed to maintain 0 mV across the tissue (i.e., Isc) continuously recorded. Transepithelial electrical resistance (Rt) was determined by measuring (every 5 min) the amount of current necessary to offset the short-circuited condition by ±3 mV for a few seconds.

The bathing medium used during the dissection and control periods of the experiments was a modified Tyrode solution with the following composition (in mM): 1.8 calcium gluconate, 1.2 MgCl2, 4.5 KCl, 103 NaCl, 30 NaHCO3, 1 NaH2PO4, 5.7 glucose, 0.3 glutathione, and 10 sucrose. The pH of this solution when bubbled with 5% CO2-95% air was 7.5. It measured ~280 mosmol/kgH2O. As needed, this solution was modified as described below. Coils of polyethylene tubing connected to a circulating water bath were placed within each hemichamber to maintain the control temperature of the bathing solutions at 36°C. For the experiments requiring adjustments in medium temperature, which was continuously monitored with a digital thermometer and kept within 0.5°C of the desired temperature, the water in the circulating bath was either heated or chilled with ice.

In separate experiments, Jdw were measured in either the tear-to-stroma or stroma-to-tear direction. Most protocols entailed the unilateral modification of bathing solution tonicity. To induce a hypertonic change to the cold side, for example, crystalline sucrose that had been predissolved into the 2 ml of replacement solution was introduced after taking the final control sample, so that on injecting this replacement, the sucrose was diluted into the hemichamber at the final concentration tested. In experiments in which the sucrose was added to the labeled side (after the final control sample), 4 ml of the "hot-side" bath was removed and added to a vial containing the appropriate mass of sucrose, which was then forcefully mixed within a warm water bath so that the crystals dissolved in ~2 min, and the final solution was returned to the labeled-side bath. With either of these approaches, the effects of sucrose concentrations on Jdw were ascertained with levels as high as 400 mM, which also caused a volume increase within the hemichamber. This increase (of ~8% in the case of 400 mM sucrose, as happens when the appropriate amount of sucrose is added to a beaker of water) was corrected for in the calculation of Jdw.

To produce hypotonic shifts, which were done unilaterally in separate experiments on either the tear or stromal sides of the preparations after the control or baseline periods, the medium in one hemichamber was replaced with a Tyrode solution that had been diluted threefold with water and to which 5 mM Tris base (Trizma, Sigma, St. Louis, MO) had been added, along with sufficient Ca2+, Mg2+, and K+ salts so that the concentrations of these electrolytes were not reduced within the chamber. This medium had a calculated osmolality of 118.9 mosmol/kgH2O and a measured osmolality of ~110 mosmol/kgH2O.

In some protocols, the control tonicity (~280 mosmol/kgH2O) was restored during the final sample periods (typically 4 samples comprising an hour of elapsed time). For this, the volume of the hemichamber containing the anisotonic medium was reduced in half and the remaining solution was rapidly superfused with 4–5 volumes of control Tyrode solution, followed by the restoration of the complete chamber volume; in cases in which this protocol was done on the hot side, sufficient 3H2O was then added to restore the baseline specific activity, which was confirmed by immediately sampling (25 µl) of the hot-side bath. Because this maneuver took several minutes, and the sampling interval of 15 min was maintained throughout the duration of the experiments, the first cold-side sample after such "washouts" underestimated the fluxes, and this value was not included in the average of the samples representing the experimental condition (for example, see Fig. 1).


Figure 1
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  		Fig. 1. Effect of 400 mM tear-side sucrose on unidirectional water fluxes (Jdw): calculated Jdw vs. elapsed duration of experiments. Each series of points represents the data obtained from an individual conjunctival preparation isolated in an Ussing-type chamber and bathed bilaterally with normal Tyrode solution (280 mosmol/kgH2O). After collection of the control samples, the tonicity of the tear-side medium was sequentially increased and restored by unilaterally adding and removing 400 mM sucrose. The first data point immediately following the "washout" to the control solution on the tear side (at t = 135 min) was not plotted, as explained in MATERIALS AND METHODS.

 
Diffusional water fluxes were also measured under Na+-free conditions in some experiments. For these, the Na+ salts of the normal Tyrode solution were replaced by 30 mM choline bicarbonate and 103 mM N-methyl-D-glucamine plus 103 mM HCl.

Mannitol fluxes. For another assessment of the integrity of the preparation (in addition to Rt measurements) under hypertonic conditions, mannitol fluxes were also measured. For these experiments, the 10 mM sucrose of the control Tyrode solution was replaced by 10 mM unlabeled mannitol, and [D-14C]mannitol was added to the apical side hemichamber (~1 µCi/ml bathing solution) at t = 0 of the flux protocol. Samples (2 ml) were then taken from the unlabeled bath and replaced with fresh solution at 15-min intervals for three experimental conditions comprising a 3-h duration. During the course of the experiment, the labeled side was sampled (25 µl) periodically to quantify the specific activity. All samples were mixed with a modified Bray solution, and the beta emission was counted in a liquid scintillation detector.

Butanol fluxes. To examine whether or not changes in bath tonicity affected the geometry of the tissue, and hence the flow of water across the lipid bilayer, fluxes of the highly lipophilic molecule n-butanol were measured. For these experiments, 1 mM unlabeled n-butanol was introduced bilaterally to the bathing solutions (a treatment that did not affect the electrical parameters), followed by the addition of [n-14C]butanol (~1 µCi/ml bathing solution) to the apical hemichamber. The flux samples were processed as described above for radiolabeled mannitol.

Definition of coefficients. The diffusion permeability coefficient Pdw is related to Jdw by the equation Pdw = Jdw/(A·Vw·Cw), where A is the area of the membrane (cm2), Vw is the partial volume of water (cm3/mol), Cw is the concentration of water (mol/cm3), and Jdw is the measured flux (cm3/s). In our analysis, Jdw has the meaning of a unidirectional flux that can cross the membrane by any available pathway. This equation was also used to calculate changes in Pdw as a function of bath temperature for the construction of Arrhenius plots.

Data analysis. The significance of experimentally elicited changes in water diffusion rates were analyzed with Student's t-test as paired data, with {alpha} = 0.05 chosen as the level of confidence.

Chemicals. Tritiated water was purchased from ICN Biochemicals (Costa Mesa, CA). [n-1-14C]butanol was ordered from American Radiolabeled Chemicals (St. Louis, MO). All other chemicals, including [D-1-14C]mannitol, were obtained from Sigma. Amiloride was prepared and stored at 5°C in aqueous solution (0.1 M), and bumetanide was also kept at refrigerator temperature as a 0.02 M stock in 95% ethanol.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Earlier work showed a reduction in Jdw across the conjunctiva when the tonicity of either the apical or stromal side baths was lowered to ~110 mosmol/kgH2O, changes that occurred with a reduction in Rt and an approximate doubling of mannitol permeability (5). The present study extended the previous work on the conjunctiva by initially examining the effects of hypertonic solutions on Jdw.

Effects of tear-side hypertonicity under control conditions. Adding sucrose to the apical bath reduced Jdw and the electrical parameters Isc and Rt, suggesting a downregulation of membrane water permeability and possible closing of epithelial conductances (reducing Isc), as well as a putative disruption in the paracellular tight junctions given the reduction in Rt (Table 1). On application of 200 mM sucrose, the control diffusional water fluxes declined ~18% from 4.29 to 3.52 µl·min–1·cm–2. The tonicity increase simultaneously eliminated half of the Isc, a decline accompanied by a 14% loss in Rt. On doubling of the sucrose concentration to 400 mM, Jdw sequentially declined an additional 16% to 2.96 µl·min–1·cm–2, so that the water fluxes were 31% lower than the control level. The epithelium responded to the increased sugar concentration with a further approximate halving of the Isc and little additional change in Rt.


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  		Table 1. Effects of hypertonic solutions on tear side of rabbit conjunctival epithelium on unidirectional water fluxes, short-circuit current, and transepithelial resistance

 
Overall, it can be noted that there is no correlation between the magnitude of the control values for Jdw and the control electrical values (Table 1). Furthermore, the individual preparations differed in the extent to which the water fluxes responded to the increased tonicity. Experiment 12 (Table 1), for example, exhibited the lowest control value for Jdw within the set, and this parameter was unaffected by the application of the sugar, although its electrical values were typical for conjunctival epithelial preparations isolated within Ussing-type chambers.

Moreover, it was observed that the addition of sucrose produced a gradual Jdw decline, with the largest changes measured between 45 and 60 min after application of the sugar. This is illustrated (Fig. 1) with data from a separate set of experiments in which 400 mM sucrose was directly introduced to the tear-side bath, followed by a washout of the apical hemichamber with control solution to restore the baseline tonicity. In these experiments, 3H2O was added apically at t = 0, and samples of the stroma-side medium were taken every 15 min. The calculated flux of the labeled water (expressed in µl·min–1·cm–2) is plotted as a function of the elapsed time of the experiment. After the fourth control sample was drawn, sucrose was added into the apical solution and four additional samples were taken before restoring the control tonicity. Considering only the last two periods with high sucrose (i.e., samples taken at 105 and 120 min), Jdw for the seven experiments averaged 2.91 ± 0.07 µl·min–1·cm–2, a flux rate 25% below the control value of 3.90 ± 0.04 µl·min–1·cm–2 (mean of the 4 control periods for each of the 7 preparations). After the removal of the 120-min sample, the apical medium was replaced with normal Tyrode solution and the specific activity of 3H2O was restored. Consequently, the next sequential sample at t = 135 min represented a flux period of <15 min, thereby resulting in an underestimation of the calculated flux so that this value was not included in Fig 1. Nevertheless, this sample served as the baseline value to calculate the flux between 135 and 150 min, etc. On restoration of the normal tonicity, Jdw recovered to 3.75 ± 0.01 µl·min–1·cm–2, or within 96% of the control rate when only the last two-sample periods for each of the seven conjunctivae are averaged. Interestingly, as shown in Fig. 1, the higher the control level of water movement (presumably a reflection of a higher intrinsic water permeability of the epithelium), the greater the reduction in Jdw in response to apical hypertonicity.

Effects of stromal side hypertonicity under control conditions. In contrast, the addition of sucrose to the stromal side bath (Table 2) did not elicit significant reductions in Jdw or marked electrical effects, even with concentrations as high as 400 mM (higher levels were not tested). Plots (not shown) of the 18 control values for Jdw compiled in Tables 1 and 2 vs. Isc and vs. Rt during the control periods did not indicate a linear correlation between the water fluxes and these electrical parameters by a least-squares method of analysis (R2 < 0.04), a persuasive indication of the independence of the unidirectional water fluxes and rates of electrolyte transport.


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  		Table 2. Effects of hypertonic solutions on stromal side of rabbit conjunctival epithelium on unidirectional water fluxes, short-circuit current, and transepithelial resistance

 
Effects of stromal side hypertonicity under Na+-free conditions. Because the epithelium did not detect the elevated tonicities applied from the stromal side of the tissue (although in earlier work it detected unilateral tonicity reductions to ~110 mosmol/kgH2O; Ref. 5), we reasoned that perhaps the apparent downregulation of Jdw occurred secondarily to changes in cell volume. Putatively, when present in the stromal side bath, sucrose may diffuse slowly through the relatively thick stroma so that its concentration in the lateral spaces may rise in a manner sufficiently limited that intrinsic regulatory volume increase (RVI) mechanisms could come into play and maintain cell volume. If so, the apparent downregulation of membrane Pdw would not occur under conditions with an adequate RVI response but would do so under conditions with which RVI mechanisms were inhibited. To test this possibility, experiments under Na+-free conditions were done, because of the importance of the cation to acute RVI mechanisms (14, 15). The bilateral removal of Na+ from the solutions bathing the conjunctiva did not significantly affect Jdw, with control values of 5.37 ± 0.53 and 5.22 ± 0.41 µl·min–1·cm–2 in the absence of Na+ (n = 4, P > 0.68, as paired data; individual values not shown for simplicity), although the typical effects of an elimination of the Isc and an increase in Rt (28) were observed. In another set of experiments, conjunctivae were mounted directly in Na+-free solution and preequilibrated with such for 1 h before introduction of 3H2O to measure Jdw (Table 3). Under these conditions, the introduction of stromal side sucrose resulted in a ~35% reduction in Jdw (from 4.09 to 2.66 µl·min–1·cm–2), which recovered by 32% (to 3.52 µl·min–1·cm–2) on restoration of the control tonicity on the stromal side with the Na+-free Tyrode solution (Table 3). Thus without Na+, and putative RVI activity, diffusional water movement was responsive to stromal side hypertonicity and to the reimposition of physiological osmolality. Although the addition of 400 mM sucrose to the stromal bath under Na+-free conditions elicited a reduction in Jdw, stromal side introduction of the sugar did not affect the residual Isc (near zero) or the Rt of 1.64 ± 0.32 k{Omega}·cm2 (n = 4) recorded in the absence of Na+.


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  		Table 3. Effect of hypertonic solution on stromal side of rabbit conjunctival epithelium on unidirectional water fluxes under Na+-free conditions

 
Effects of stromal side hypertonicity in presence of amiloride and bumetanide. To further examine the possible link between presumptive cell volume changes and Jdw, 400 mM sucrose was added to the stromal baths of conjunctivae preexposed to amiloride and bumetanide, inhibitors of Na+/H+ exchange activity and the Na+-K+-2Cl– cotransporter, respectively, which mediate RVI mechanisms in most epithelia (14, 15) and are present in the conjunctiva (32, 33). As in the case of introducing a Na+-free medium, the simultaneous addition of the two inhibitors to the stromal bath did not affect Jdw; however, the sequential introduction of 400 mM stromal side sucrose reduced the H2O fluxes by ~20% (from 3.84 to 3.08 µl·min–1·cm–2; Table 4, protocol A) With conjunctivae pretreated with the inhibitors (Table 4, protocol B), the fluxes were reduced by 31% on application of stromal side sucrose (from 4.67 to 3.22 µl·min–1·cm–2), and recovered by a significant 17% (to 3.76 µl·min–1·cm–2) on restoration of the control tonicity to the stromal hemichamber while maintaining the presence of the inhibitors. As in the case of the Na+-free conditions, the addition of 400 mM stromal side sucrose did not affect the Isc and Rt of tissues preexposed to amiloride and bumetanide, although the combination of these inhibitors eliminated about two-thirds of the Isc without a significant effect on Rt (n = 8).


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  		Table 4. Effect of hypertonic solution on stromal side of rabbit conjunctival epithelium on unidirectional water fluxes in presence of amiloride plus bumetanide

 
Effects of tear-side hypertonicity on mannitol fluxes. It was demonstrated earlier (5) that the reduction in Jdw across the conjunctiva in response to basal hypotony coincided with an approximate doubling of the mannitol permeability, indicating that the unilateral hypotonic conditions had adversely affected the integrity of the paracellular pathway and that the reduction in diffusional water movement most likely occurred because of a reduced transcellular movement. For comparative purposes, the effects of 400 mM tear-side sucrose on mannitol fluxes were determined. The addition of the sugar (which decreased water fluxes) increased the mannitol flux (Table 5) and the calculated mannitol permeability by ~39%, an indication that the paracellular pathway was not involved in the water flux reduction. Return to the control tonicity at the end of the experiments, however, resulted in a substantial, threefold increase in mannitol permeability and a decline in Rt (to 36% of the control level; Table 5), implying that an undetermined portion of the recovery of Jdw on restoration of the control conditions might involve H2O movement via the paracellular pathways.


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  		Table 5. Effects of sucrose addition to the tear-side bath of rabbit conjunctivae on transepithelial mannitol fluxes and resistance

 
Effects of glutaraldehyde on Jdw under hyper- and hypotonic conditions. To examine the prospect that the tonicity-evoked changes in Jdw reflect dynamic cellular responses, the effects of glutaraldehyde were determined. For this, Jdw was measured across conjunctival preparations for a 1-h period, after which 1.2% glutaraldehyde was bilaterally applied and maintained in the bathing solutions to preserve the tissue in vitro (9). The addition of the fixative, in itself, did not significantly affect Jdw, which was 5.48 ± 0.68 µl·min–1·cm–2 under control conditions and 5.24 ± 0.57 µl·min–1·cm–2 in the presence of 1.2% glutaraldehyde (n = 10, P > 0.16, as paired data; Table 6, combined data from protocols A and B). After 1 h of sampling in the presence of 1.2% glutaraldehyde, 400 mM sucrose was introduced to the apical hemichamber (Table 6, protocol A). Under these conditions, the hypertonic bath did not affect Jdw, and the fluxes were not increased on restoration of the control tonicity (glutaraldehyde maintained during the washout of the apical chamber).


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  		Table 6. Effects of unilateral anisotonic conditions on unidirectional water fluxes across rabbit conjunctivae preexposed to glutaraldehyde

 
As to the electrical parameters, the addition of 1.2% glutaraldehyde rapidly inhibited the Isc (the current went to 0 within 2–3 min; n = 10, data not shown) without a significant effect on Rt, which was 0.74 ± 0.08 k{Omega}·cm2 under control conditions and 0.71 ± 0.07 k{Omega}·cm2 in the presence of glutaraldehyde (n = 10, P > 0.24, as paired data). The electrical results suggest that conformational changes associated with rheogenic transporters and the Na+-K+ pump were inhibited by the aldehyde, whereas the ionic channels may have been preserved in their control state.

Because the application of apical hypotonic conditions also reduces Jdw, as described previously (5), the effects of 1.2% glutaraldehyde on this opposite tonicity challenge were also determined. As shown in Table 6, protocol B, there was no significant effect of hypotonicity in the presence of the fixative. Restoration of the control osmolality in the presence of glutaraldehyde led to an approximate 8% decline in Jdw but not the recovery observed previously (5), which was confirmed in a set of four experiments that were done without glutaraldehyde in tandem with those shown in Table 6. In these additional experiments, the control value for Jdw was 3.70 ± 0.55 µl·min–1·cm–2, and Jdw was about 18% lower, or 3.02 ± 0.40 µl·min–1·cm–2, when the apical hemichamber contained the 110 mosmol/kgH2O solution (n = 4, P < 0.05, as paired data); on restoration of the control Tyrode solution to the apical hemichamber, Jdw recovered to ~95% of the control level (n = 4, P < 0.05). Thus glutaraldehyde prevented changes in Jdw typically seen on altering tear-side tonicity.

However, a reduction of Jdw in response to basal hypotony was not prevented by 1.2% glutaraldehyde. When conjunctivae pretreated bilaterally with 1.2% glutaraldehyde were exposed to 110 mosmol/kgH2O solution on the stromal side, the water fluxes declined from 4.49 ± 0.72 to 3.54 ± 0.48 µl·min–1·cm–2 (n = 6, P < 0.05, as paired data), a 21% change similar to that observed previously (5). Furthermore, on restoration of the control tonicity to the stromal side, the fluxes recovered to within 6% of the control value (or to 4.23 ± 0.72 µl·min–1·cm–2; n = 6, P < 0.05), although the 1.2% glutaraldehyde was maintained bilaterally. Additional experiments determined that a concentration of 2% glutaraldehyde for 3 h was necessary to preclude an effect by basal hypotony on Jdw. In these experiments, 2% glutaraldehyde was bilaterally added to the chambers of freshly mounted conjunctivae, followed 2 h later by addition of 3H2O to the apical bath. After an additional hour of sampling to determine the baseline flux (Table 6, protocol C), the tonicity of the stromal side hemichamber was reduced to 110 mosmol/kgH2O, a maneuver that did not elicit a significant effect on Jdw (P > 0.29, as paired data); nor were the fluxes notably affected on restoration of the control osmolality (P > 0.22, as paired data).

In contrast, a striking decline in Rt was recorded on restoration of the control osmolality. For the 10 tissues treated with the 1.2% concentration (Table 6, protocols A and B), the mean Rt during the anisotonic periods of 0.48 ± 0.03 k{Omega}·cm2 was reduced to 0.25 ± 0.01 k{Omega}·cm2 on restoration of the control tonicity (n = 10, P < 0.01). Similarly, in the four experiments preserved with 2% glutaraldehyde (Table 6, protocol C), restoration of the control tonicity produced an Rt loss from 0.39 ± 0.04 to 0.21 ± 0.03 k{Omega}·cm2 (n = 4, P < 0.05). Therefore, whereas the washout protocol to reintroduce the control medium adversely perturbed Rt across the fixed conjunctivae, Jdw did not increase (Table 6, final column of all protocols), suggesting the absence of significant water movement via the paracellular pathways.

Effects of anisotonic conditions on butanol fluxes. To assess the possibility that the anisotonic conditions reduced water movement across the lipid bilayer because of putative alterations of tissue geometry in vitro, transepithelial fluxes of n-butanol, a highly lipophilic compound (12, 13), were measured. For these experiments, after 1 h of sampling with normal Tyrode solution, the tonicity of the apical side bath was increased by the addition of 400 mM sucrose for an additional hour of sampling, after which the tonicity of the stromal side compartment was reduced to ~110 mosmol/kgH2O. Thus during the third sampling hour, a tonicity gradient of ~680–110 mosmol/kgH2O existed across the tissue in the tear-to-stroma direction. Nevertheless, these inordinate conditions did not significantly affect transepithelial [n-14C]butanol movement (Table 7, protocol A). In contrast, Jdw was successively inhibited on the sequential introductions of the tear-side hypertonic and stroma-side hypotonic states (Table 7, protocol B). The water fluxes in the presence of the ~680–110 mosmol/kgH2O gradient (2.22 µl·min–1·cm–2) were 38% below the control value and partially recovered (to 3.06 µl·min–1·cm–2) to within 15% of the control level on bilateral restoration of the normal Tyrode solution. Overall, membrane components (presumably water channels) responded to the anisotonic milieu, whereas the lipid bilayer was unaffected, based on the unaltered n-butanol fluxes.


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  		Table 7. Sequential effects of tear-side hypertonicity and stromal hypotonicity on unidirectional butanol (protocol A) and water (protocol B) fluxes across rabbit conjunctivae

 
Arrhenius plots from temperature-dependent measurements of Pdw under control and anisotonic conditions. Water fluxes were measured across conjunctival epithelia at three temperatures (typically 286K, 299K, and 312K) under control conditions; thereafter, the bath tonicity in either one or both hemichambers was altered, and Jdw measurements were repeated at the same three temperatures used to obtain the control values. In this manner, paired data were acquired to construct Arrhenius plots for the effects of tear-side hypertonicity with 400 mM sucrose (Fig. 2, plot A; n = 6 conjunctivae), stroma-side hypotonicity (Fig. 2 plot B; n = 4), and the combination of tear-side sucrose plus stromal hypotonicity (Fig. 2, plot C; n = 6). The actual temperatures applied within each of these experimental sets (Fig. 2, sets A–C) were consistent within each set so that means and SEs could be calculated. The slopes of the plots of ln Pdw vs. 1/KR represent the activation energy (Ea) for water movement across the epithelium; a comparatively greater slope depicting a relatively higher Ea level implies that proportionally more transepithelial diffusion is occurring across the lipid bilayer, that is, there is less water movement through water channels (37). The results shown in Fig. 2, which includes the equation for each line generated by linear regression analysis, indicate that the anisotonic conditions led to reduced diffusion via water channels, given the steeper slope exhibited by the experimental condition compared with its respective control. On average, the control Ea for the 16 conjunctivae used to generate the data depicted in Fig. 2 was 4.93 ± 0.11 kcal/mol, a level consistent with water movement predominantly through aqueous pores (36, 37, 39). The anisotonic conditions applied in experimental sets A–C evoked respective increases in slope of 31%, 41%, and 38%, or to Ea levels that likely reflect a partial inhibition of water channel activity.


Figure 2
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  		Fig. 2. Transepithelial water fluxes were measured across conjunctival epithelia at 3 temperatures (e.g., 286K, 299K, and 312K) initially under control conditions and then repeated at the same temperatures with medium osmolality altered as indicated. Points are means ± SEs of ln diffusion permeability coefficient (Pdw) with n = 6, 4, and 6 for experimental sets A, B, and C, respectively. In each experimental set, the increase in slope elicited by the anisotonic conditions was significant as paired data (P < 0.05). Experimental Set A: effect of tear-side hypertonicity; Set B: effect of stromal hypotony; Set C: effect of tear-side sucrose plus stromal hypotony.

 
Effect of mercuric chloride on Jdw across the conjunctiva. To ascertain whether the apparent downregulation of water channel activity under anisotonic conditions (Fig. 2) could be linked to AQPs, the effects of 0.5 mM HgCl2 were evaluated. Our earlier unpublished data indicated that this level of Hg2+ did not significantly affect Jdw when applied to either side of the tissue (n = 12 conjunctivae), although the epithelium is known to express AQP3 (35), an aquaporin sensitive to mercurials. Nevertheless, experiments were directed to determine the effects of 400 mM tear-side sucrose on Jdw across conjunctivae preexposed to HgCl2. In these new experiments, the introduction of the mercurial to the tear-side bath reduced Jdw by ~6% and prevented a succeeding reduction in the water fluxes from the addition of sucrose (Table 8). Although these results are seemingly consistent with the involvement of AQPs in the hypertonicity-induced reduction in Jdw, the following caveat must be considered. The addition of Hg2+ also rapidly eliminated the Isc within 6–8 min and adversely compromised Rt (Table 8), suggesting that the well-documented cytotoxic effects of Hg2+ (17, 18, 29) might have come into play and precluded a cellular response to the anisotonic environment. Presumably, this loss of cellular viability in the presence of Hg2+ might also explain the fact that Jdw actually increased on addition of sucrose to the Hg2+-pretreated tissues (Table 8), instead of the reduction usually obtained under hypertonic conditions.


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  		Table 8. Effects on unidirectional water fluxes, short-circuit current, and transepithelial resistance of hypertonic solutions on tear side of rabbit conjunctival epithelia pretreated with mercuric chloride

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Conjunctival epithelial preparations that were isolated in a classic Ussing-type arrangement were unilaterally exposed in separate experiments to hyper- and hypotonic media interfacing with either their apical or stromal surfaces. In each of these four separate conditions, the anisotonic medium elicited a reduction in unidirectional water movement across the tissue, with the exception that to obtain such reduction in Jdw with increased tonicity from the stromal side, either Na+ must be absent from the bath or inhibitors of RVI mechanisms (amiloride and bumetanide) must be present. Overall, these observations imply that reductions in epithelial membrane water permeability apparently occur secondarily to cell volume changes and that this phenomenon could contribute to volume regulation. Hypothetically, water channels in either, or both, the apical and basolateral aspects could have closed or been removed from the membranes during the anisotonic periods. A reduction in membrane water permeability would abet RVI, as well as regulatory volume decrease (RVD) mechanisms, by reducing water flow out from, or into, the cellular compartment during respective hypertonic or hypotonic conditions. Such putative reduction in cell membrane water permeability in anisotonic media would be advantageous to cellular function by mitigating extensive volume changes.

The rabbit conjunctival epithelium expresses a Na+-dependent, bumetanide-inhibitable Cl– transport directed in the basolateral-to-apical direction, as well as an amiloride-resistant Na+ absorptive process at the apical surface (28, 32). From electrical measurements recorded at this laboratory (28), the control Rt measured across many preparations resembles that observed in a tight epithelium (>1 k{Omega}·cm2), although there is also a large variability in control Rt values, with some preparations having relatively low Rt. Presumably, paracellular conductance may increase in some cases from the mechanical placement of this rather fragile epithelium in the chamber. Consistent with this possibility, transepithelial fluxes of Na+ (27) and Cl– (28) showed that the paracellular movements of these electrolytes were about twice that of their transcellular movements and that the magnitude of these transepithelial fluxes was inversely related to Rt.

In contrast, transepithelial water fluxes seem to be predominately transcellular, because data were obtained that indicate independence between Jdw and the integrity of the paracellular pathway: 1) there is no correlation between control levels of Jdw and control Rt values (Tables 1 and 2); 2) mannitol fluxes, which solely traverse the paracellular pathway, are increased by both hypertonic (Table 5) and hypotonic (5) conditions, which reduce Jdw comparably; and 3) with tissues fixed by glutaraldehyde, restoration of the control tonicity as the final experimental condition (Table 6) led to a substantial deterioration of Rt without an increase in transepithelial water fluxes. Although it is not clear as to why Rt was adversely affected during the restoration of the control tonicity with the glutaraldehyde-treated conjunctivae (perhaps tight junctions were mechanically compromised by the washout protocol), it was nevertheless consistently found that the water permeability of these epithelia was not affected by the imposition of the anisotonic conditions or the reintroduction of the control tonicity (Table 6). Furthermore, it is interesting to note that glutaraldehyde in itself does not affect the water channels in the amphibian bladder (23), and its introduction to the conjunctiva did not have a direct, pronounced effect on Jdw (Table 6).

Others have applied glutaraldehyde in various cellular systems under conditions that produced a submaximal fixation so that vital enzymatic functions (e.g., glycolysis) could be preserved (1, 6, 20, 24). This approach generally entails brief periods of exposure (~5–20 min) with ~100-fold lower concentrations than we used in the present study. Such submaximal fixations have been implemented to "lock" the Na+/H+ exchanger of dog red blood cells, which is activated by cell shrinkage, in either the activated or inactivated state depending on the cell volume at which the fixation treatment took place (24). Similarly, the osmotic permeability of the amphibian bladder was preserved after the withdrawal of antidiuretic agonists (9, 10, 23), thereby maintaining the hormone-dependent water channels in place.

Our preliminary data indicated that the reduction in Jdw obtained under anisotonic conditions could not be prevented by limited glutaraldehyde treatments. Although glutaraldehyde is a well-known cross-linking agent of proteins that quickly reacts with amino groups (as well as various sulfhydryl- and hydroxyl-containing compounds), it penetrates relatively slowly into tissues (9). Our introduction of the agent elicited a prompt (within 2–3 min) elimination of the Isc (a current loss that occurred without an effect on Rt), thereby indicating that the fixative suppressed the activities of the Na+-K+ pump and the Na+-K+-2Cl– cotransporter, the two main elements underlying the transconjunctival Isc (28, 34). With glutaraldehyde maintained in the bathing solutions, the Jdw across the system did not respond to anisotonic conditions. Because we obtained indications (Tables 2–4) that the apparent downregulation in Pdw under stromal side hypertonic conditions may occur secondary to changes in cell volume, it is possible that the enzymatic cell signaling mechanisms by which the cell detects a change in its volume, or perhaps the cell volume itself, must be frozen by the fixative to preclude changes in Jdw. This implies that in the case of the response to stromal side hypotony (Table 6), the inhibition of the enzymatic activities of the more basal cells in the epithelium is required to prevent a change in the unidirectional water fluxes. Overall, relatively high concentrations of glutaraldehyde may have been needed because the preparations represent a multilayered epithelium upon a thick stroma.

Independently of glutaraldehyde, the restoration of the control tonicity, when done at the end of some experiments, produced substantial declines in Rt to a level just above that of the solution resistance (determined to be ~0.16 k{Omega}·cm2 under the conditions used). Yet it appears that the recovery in Jdw during this phase of these experiments (e.g., Tables 3 and 4) was not dependent on increased water diffusion via the paracellular pathways, based on the points noted above.

Adverse effects on conjunctival Rt were also conspicuous on exposure to tear-side hypotony, which produced an ~70% loss in resistance when the apical tonicity was unilaterally reduced to ~108 mosmol/kgH2O (5). Presumably, cell swelling may have affected the integrity of the tight junctions, as well as elicited increases in K+ and Cl– conductances as part of an RVD response. Conversely, declines in Rt were less pronounced (<20%) when the sucrose concentration of the apical bath was raised to 400 mM, essentially increasing the bath tonicity to ~680 mosmol/kgH2O (Table 1). Because this condition also produced a relatively limited 39% increase in mannitol permeability (Table 5), it would appear that the epithelium was more resilient to the imposed hypertonic shift than to the hyposmotic shift. Although we are proposing that membrane Pdw is downregulated under both of these anisotonic extremes to support volume maintenance, it also appears that, given the large content of Na+ and Cl– in the bathing solutions, RVI mechanisms are more readily effectuated than RVD, which requires the efflux of a relatively limited osmolyte content. This difference in the relative efficiency between RVI and RVD mechanisms that are based on electrolyte transport may explain the finding that hypertonic solutions on the stromal side did not affect Jdw (Table 2), unless presumptive RVI mechanisms were inhibited (Tables 3 and 4).

The fact that there was a decline in Jdw on addition of sucrose to the tear-side bath (Table 1) does not necessarily imply that the water permeability of only the apical surface was affected by the increased tonicity. Changes in the tonicity within the multilayered epithelium ultimately determine the water movement across the tissue and not necessarily the side to which the osmolality was altered. Nevertheless, the apical surface of the conjunctiva can be exposed naturally to virtually pure water, or strongly hyposmotic solutions, as well as elevated osmolalities in the cases of individuals with various lacrimal gland deficiencies (11). As such, the demonstrated reduction of water permeability on the imposition of anisotonic conditions in the tear-side bath seems relevant to conjunctival physiology. However, it should also be noted that we chose relatively extreme tonicity alterations to clearly obtain salient effects due to limits in the resolution of detectable changes in measurements of Jdw. It is also important to note that those who have studied cell volume regulation under anisotonic conditions (14, 15) frequently use nonphysiological osmolalities to demonstrate cellular properties and uncover latent mechanisms.

Given that the diffusive component across the lipid bilayer is proportional to the concentration of water (~55.5 M), the effects observed when solutes are changed in the decimolar range must be ascribed to the diffusive component across protein channels. The absence of an effect by anisotonic conditions on transconjunctival butanol fluxes and the increase in Ea obtained with Arrhenius plots are consistent with this. Data from the latter approach suggest that most of the transepithelial water diffusion occurs via water-transporting channels presumably at both surfaces and perhaps, as well, through the communicating junctions within the multilayered epithelium. This is based on the finding that the control Ea for water diffusion of 4.93 kcal/mol resides between the value of 2.7 kcal/mol reported for isolated endosomes containing the water channels of the anuran urinary bladder (36) and a value of 10.2 kcal/mol obtained with native Xenopus oocytes (39), which naturally exhibit relatively low intrinsic water permeability. The approximate 30–40% increase in Ea calculated under anisotonic conditions (Fig. 2) implies a partial reduction in water movement by channel-mediated pathways in the conjunctiva.

However, we have not yet identified the specific channels that are "downregulated" by nonphysiological osmolalities. Our use of HgCl2 as an agent to implicate the involvement of AQPs in the observed changes in Jdw yielded inconclusive results. This compound at concentrations 1 and 2 orders of magnitude lower than those used to inhibit water movement in oocytes (21) affects cell viability because of its reactivity with numerous biological ligands (17, 18, 29). It is possible that the millimolar levels of the mercurial produced a necrosis within the conjunctival epithelium (17, 18, 29). Any putative AQP blockade in the conjunctiva by Hg2+ appears to have been supplanted by water movement via undefined aqueous pathways across the compromised tissue.

Thus the possibility that AQPs may be involved in the observed reductions in Jdw evoked by anisotonic conditions remains an open question. Presently, only AQP3 has been confirmed in the basolateral membranes of the conjunctiva (35). Very recent data from expression microarray assays (31) indicate message for AQP5 in the human conjunctiva (personal communications from H. Turner and M. Wolosin). Assuming functional expression of these channels in the rabbit conjunctival membranes, such moieties could be present in the apical membrane, because this AQP is regarded as an apical homolog (16, 19), with trafficking properties regulated by cAMP (30). Future work could examine the hypothesis that a downregulation of epithelial water permeability occurs as a consequence of either water channel gating or trafficking. Indirect evidence consistent with a possible gating mechanism regulated by PKC and dopamine in the case of AQP4 has been described (38).

Alternatively, changes in the activity of ionic channels that are known to exhibit relatively high water permeabilities could underlie the reductions in water fluxes evoked by osmolality changes. CFTR might be a candidate in this regard given that its reported water permeability is on the order of that seen with AQP3 (25). In addition, some classes of K+ channels also appear to have equally high water permeabilities as the aquaporins (25).

In short, because we have also found decreases in diffusional water movement under anisotonic conditions across the amphibian bladder and cornea (2, 3), changes in water permeability in response to osmotic challenge may represent an unrecognized epithelial property that contributes toward cell volume regulation.


   ACKNOWLEDGMENTS
 
This work was supported by National Eye Institute Grants EY-00160, EY-01867, and EY-15857 and by an unrestricted grant from Research to Prevent Blindness, Inc., New York, NY.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. A. Candia, Dept. of Ophthalmology, Mount Sinai School of Medicine, 100th St. and 5th Ave., New York, NY 10029 (e-mail: oscar.candia{at}mssm.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.


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