Swelling-Activated Cl⁻ Channels Support Cl⁻ Secretion by Bovine Ciliary Epithelium

Abstract

purpose. To determine whether swelling-activated Cl⁻ currents (I_Cl,swell) observed in isolated nonpigmented ciliary epithelial (NPE) cells contribute to Cl⁻ secretion across the ciliary epithelium.

methods. Ion transport across intact bovine ciliary epithelium was monitored electrically. Native isolated bovine NPE cells were harvested enzymatically. Cell volume changes were measured by calcein-fluorescence quenching.

results. Bilateral reduction in osmolality transiently increased short-circuit current (I_sc), averaging 60% to 70%. Bilateral pretreatment with 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), a Cl⁻ channel blocker, reduced I_sc stimulation by ∼60%, suggesting that transcellular I_Cl,swell largely mediates the increased current. The hypotonically-triggered I_sc stimulation was also inhibited by phloretin, a blocker of swelling-activated Cl⁻ channels and by flufenamic acid, a blocker of Cl⁻ and nonselective cation channels. Cyclamate substitution for bath Cl⁻ reduced the baseline I_sc and the increase in hypotonically-triggered I_sc. In that case, addition of either NPPB or flufenamic acid did not produce further inhibition. The transepithelial responses were correlated with regulatory volume responses of freshly harvested NPE cells. Hypotonicity elicited a regulatory volume decrease (RVD) over a period comparable to that of the hypotonicity-triggered increase in I_sc. The RVD was also inhibited by Cl⁻-channel blockers and by Cl⁻ substitution.

conclusions. I_Cl,swell of NPE cells is functionally expressed in intact ciliary epithelium and is oriented to subserve aqueous humor formation. NPE cell volume can be measured with calcein-fluorescence quenching. I_Cl,swell may be stimulated by increased stromal fluid uptake and delivery to the NPE cells, facilitating Cl⁻ secretion and increasing fluid release into the posterior chamber.

The formation of aqueous humor is driven primarily by NaCl secretion across the ciliary epithelium (Fig. 1) , thereby establishing an osmotic gradient for water movement. ^{1 2 3 4 5 6} The bilayered ciliary epithelium covers the ciliary body and comprises an outer pigmented (PE) and an inner nonpigmented (NPE) cell layer. Intercellular gap junctions link cells both within and between the PE- and NPE-cell layers. ⁷ Solute, primarily NaCl, is taken up from the stroma of the ciliary processes by the PE cells, transferred to the NPE cells through gap junctions, and finally released into the aqueous humor of the posterior chamber. ^{8 9 10 11 12}  

The efflux of Cl⁻ through anion channels of NPE cells plays a central role in forming the aqueous humor, and the rate of this Cl⁻ release may limit the rate of aqueous humor secretion. ^{5 13 14 15} Our understanding of these Cl⁻ channels is largely derived from electrophysiologic and volumetric measurements of hypotonically-perfused isolated NPE cells. The focus on hypotonic perfusion has stemmed from two considerations. First, hypotonic cell swelling provides a convenient probe of Cl⁻ channels in these cells. Swelling activates readily measurable Cl⁻ currents (I_Cl,swell), ^{16 17 18 19 20 21} thereby facilitating electrophysiologic characterization. Second, I_Cl,swell may play a physiologic role in the net formation of aqueous humor. Transfer of NaCl and water from the PE cells is expected to increase the volume of the NPE cells, thereby triggering I_Cl,swell at the aqueous surface and enhancing secretion. ²² A physiologic role has also been suggested for I_Cl,swell of the PE cells, increasing release of Cl⁻ back into the stroma and thereby reducing net aqueous formation (Fig. 1) . ⁴  

I_Cl,swell of ciliary epithelial cells has also been studied volumetrically. Cell swelling triggers K⁺, as well as Cl⁻, release into the extracellular solution, ^{8 12 13 21} with water passively following the release of solute. The release of fluid to the extracellular space shrinks the cell, restoring cell volume toward its control isotonic level. This response to hypotonic challenge is termed the regulatory volume decrease (RVD). ²³ The relevance to aqueous humor formation of these studies of hypotonically-perfused isolated cells depends on the tacit assumptions that swelling-activated Cl⁻ channels contribute significantly to Cl⁻ secretion across the native ciliary epithelium and that the time courses are comparable for both events triggered by hypotonicity, the putative increase in transepithelial Cl⁻ secretion and the RVD. The validity of these assumptions is central to current views of aqueous humor formation and depends on the unknown vectorial distribution of Cl⁻ channels within the ciliary epithelium. The tight junctions linking neighboring NPE cells separate the NPE basolateral channels facing the aqueous surface from the channels at the NPE apical surface and the channels of the entire PE surface which face the stroma (Fig. 1) . Depending on the relative number and activity of the two topologically defined populations of Cl⁻ channels, swelling activation may increase, decrease, or have no effect on net Cl⁻ secretion. To the best of our knowledge, these assumptions have never been tested. The purposes of the present study were to determine the contribution of I_Cl,swell toward Cl⁻ secretion across the bovine ciliary epithelium and to learn whether the putative effect is expressed over a period comparable to that of the RVD. Fluid release from the NPE cells was followed with a recently described calcein-fluorescence quenching technique ^{24 25} for monitoring volume of isolated cells. 

Methods

Preparation of Bovine Ciliary Epithelium and NPE Cells

As described previously, ²⁶ fresh bovine eyes were obtained from a local abattoir. For the transepithelial measurements, a sector of fresh bovine ciliary epithelium with supporting stroma was excised and mounted in an Ussing chamber to monitor the transepithelial currents. ^{5 27 28} For the volumetric measurements, dissociated native bovine NPE cells were obtained by enzymatic digestion. In brief, ciliary processes were excised and rinsed with Dulbecco’s phosphate-buffered saline (PBS; Invitrogen-Gibco, Grand Island, NY). The dissociated cells were obtained by incubating the preparation with 0.25% trypsin in a shaker for 30 to 35 minutes (250 rpm) at 37°C. The cells were washed twice with PBS and plated on coverslips in medium 199 for at least 2 hours at 37°C in 5% CO₂ before the experiment. The medium contained 10% fetal bovine serum and 0.1% gentamicin (Invitrogen-Gibco). For both measurements, the bovine preparations were prepared approximately 1.5 hours after death. 

Transepithelial Electrical Measurements

The bovine ciliary epithelium was mounted in a custom-built Ussing chamber connected to a water reservoir. The area of ciliary epithelium exposed to the bathing solution was 0.35 cm². Both sides of the chamber were filled with 15 mL isotonic Ringer’s solution bubbled continuously with 5% CO₂/95% O₂. Drugs were added to the bathing solutions when the measured transepithelial potential difference (PD, aqueous surface with respect to stromal surface) and tissue resistance (R), and calculated short-circuit current (I_sc, positive current from stroma to aqueous humor surface) became stable. By “I_sc,” we refer to the current necessary to reduce the spontaneous PD to zero, even in the presence of osmotic asymmetries across the tissue. We verified early in the study that the values of calculated I_sc and measured I_sc were the same. Experiments were conducted at room temperature. 

Spontaneous PD and voltage responses (ΔPD) to current pulses (ΔI) were recorded with two pairs of Ag/AgCl electrodes, EKC for current- and EKV for voltage-sensing electrodes (World Precision Instruments, Sarasota, FL). The electrodes were connected to a voltage–current clamp (DVC-1000; World Precision Instruments). The analog data were digitized and stored in a computer with a digitizer (Quad-16) coupled with computer software (Data-Trax; World Precision Instruments). The R and I_sc were calculated as (ΔPD/ΔI) and (PD/R), respectively. ⁵  

Cell Volume Measurements

Cell volume was measured continuously by monitoring the level of calcein fluorescence. As previously described, ^{26 29} coverslips were mounted in a chamber connected to a microscope (Diaphot; Nikon, Tokyo, Japan). Cells were loaded with 2 μM calcein-AM and 0.02% Pluronic for 30 to 40 minutes, and subsequently superfused with isotonic Tyrode’s solution for 30 minutes before data acquisition at room temperature. To minimize bleaching, calcein was excited every 20 seconds at 488 nm, and light emitted at 520 nm was detected with an IC-200 charge-coupled device (CCD) camera (Photon Technology International, Princeton, NJ). Data analysis was conducted with Imagemaster software (Photon Technology International). 

The measurements of NPE-cell volume were conducted with dissociated cell preparations that included both NPE and PE cells. The PE cells are readily identified by their prominent cell granules, which has permitted investigators to measure and differentiate the electrophysiologic and volume-regulatory properties of the two cell types, even in mixed harvested populations. ^{20 26 30}  

In previous studies of a small amount of ocular cells, we ^{26 29} and others ¹² have taken cell area as an index of cell volume. We have relied on indirect approaches to distinguish between shrinkage and contraction. ^{26 29} A more fundamental approach to this complexity is provided by monitoring total calcein fluorescence, whose quenching at intracellular sites is enhanced by shrinkage and reduced by swelling but unaltered by changes in cell shape. Calcein-fluorescence quenching was initially proposed as a technique for measuring cell volume by Hamann et al., ²⁴ and fully validated recently for cultured astrocytes. ²⁵ The volume-dependent calcein quenching has been demonstrated not to reflect quasi-confocal effects, self-quenching or interaction with NaCl. ²⁵ The precise mechanism is not entirely clear, but apparently arises in part from interaction with intracellular macromolecules ²⁵ and intracellular iron. ³¹  

Solutions and Pharmacologic Agents

For the transepithelial measurements, the isotonic HCO₃ ⁻-rich Ringer’s solution contained (in mM): 85.0 NaCl, 4.6 KCl, 21.0 NaHCO₃, 0.6 MgSO₄, 7.5 d-glucose, 1.0 glutathione (reduced form), 1.0 Na₂HPO₄, 10.0 HEPES, 56.0 d-mannitol and 1.4 CaCl₂ (∼295 mOsM/kg H₂O). The pH was adjusted to 7.4 after bubbling with 5% CO₂/95% O₂. The hypotonic solution (∼225 mOsM/kg H₂O) was prepared by omitting the d-mannitol. In low-Cl⁻ (12.4 mM) solution, Cl⁻ was replaced by an isosmolar amount of cyclamate ion; the residual Cl⁻ ensured well-defined half-cell potentials at the Ag/AgCl electrode surfaces. 

For the cell volume measurements, the isotonic Tyrode’s bathing solution contained (in mM): 110.0 NaCl, 15.0 HEPES, 2.5 CaCl₂, 1.2 MgCl₂, 4.7 KCl, 1.2 KH₂PO₄, 30.0 NaHCO₃, and 10.0 glucose (∼300 mOsM/kg H₂O). Solutions were made 25% (∼232 mOsM/kg H₂O) and 50% (∼160 mOsM/kg H₂O) hypotonic by reducing the concentration of NaCl from 110 to 70 and 30 mM, respectively. Solutions were rendered 25% (∼382 mOsM/kg H₂O) and 50% (∼462 mOsM/kg H₂O) hypertonic by adding 78 and 155 mM mannitol, respectively. Cl⁻-free solutions were prepared by replacing Cl⁻ by methylsulfonate. The pH was maintained at 7.35 for all solutions. 

All chemicals were reagent grade. Phloretin, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), and flufenamic acid were obtained from Sigma-Aldrich (St. Louis, MO). Pluronic F-127 and calcein-AM were purchased from Invitrogen (Eugene, OR). All agents were dissolved in dimethyl sulfoxide (DMSO) before adding to the bathing solution. The final concentration of DMSO in the solution was less than 0.1%. 

Statistical Analysis

Results are presented as the means ± SEM. Statistical significance was tested by Student’s t-test (paired and unpaired). P < 0.05 was considered significant. 

Results

Transepithelial Current Measurements: Effects of Bilateral Hypotonicity

Consistent with previous reports, ^{5 27 28} the aqueous surface was negative with respect to the stromal surface of isosmotically bathed bovine ciliary epithelium (PD = −3.5 ± 0.1 mV, R = 153 ± 2 Ω · cm², n = 253). The corresponding I_sc was 23.4 ± 0.6 μA · cm⁻², in a direction consistent with net Cl⁻ secretion from the stromal to aqueous solution. 

As illustrated by Figure 2A , reducing the osmolality of both the stromal and aqueous bathing solution by 25% produced a transient increase in PD. Thereafter, the voltage spontaneously declined toward the baseline value. Restoration of isotonicity elicited the opposite effect, transiently lowering PD. R, however, remained constant under both isotonic and hypotonic conditions. Therefore, the increase in PD elicited a proportionate change in I_sc (Fig. 2B) . In a series of 33 preparations, the baseline I_sc bathed in isotonic solution was 23.7 ± 1.5 μA · cm⁻². Addition of hypotonic solution to both sides increased the I_sc to a peak of 38.3 ± 2.4 μA · cm⁻². When the individual peak I_sc increase was normalized with respect to the baseline I_sc, the mean percentage stimulation was 64% ± 4% (P < 0.001, n = 33). R before and after hypotonic challenge was 159 ± 7 and 160 ± 7 Ω · cm², respectively. Because preparations with low baseline I_sc demonstrated variable responses on hypotonic treatment, those preparations with initial I_sc smaller than 12 μA · cm⁻², comprising 10 to 15% of the total, were excluded from the data analysis. Generally, the percentage change of I_sc increase remained relatively constant over a range of baseline I_sc from 12 to 50 μA · cm⁻². 

Effects of Cl⁻ Channel Blockers and Reduced Bathing Cl⁻ Concentration on Responses to Bilateral Hypotonicity

The Cl⁻-channel blocker (NPPB, 100 μM) is known to produce an inhibition of baseline I_sc, suggesting that the I_sc is carried by Cl⁻. ⁵ In the present study, brief pretreatment with the Cl⁻-channel blocker NPPB (100 μM) bilaterally in isotonic solution for 15 minutes reduced the subsequent hypotonically-triggered increase in I_sc (Fig. 3) . In the presence of bilateral NPPB, the mean I_sc increase was 6.5 ± 1.4 μA · cm⁻² on hypotonic treatment (n = 12). The initial mean I_sc, however, was similar between control (23.7 ± 1.5 μA · cm⁻²) and NPPB-pretreated preparations (23.9 ± 2.4 μA · cm⁻²). We tested the significance of this inhibition by comparing the response of the hypotonically-triggered I_sc increase between control and NPPB-pretreated preparations. In comparison with an increase in I_sc of ∼15 μA · cm⁻² in the control group, pretreatment of NPPB produced a mean inhibition of 59% ± 8% (n = 12, P < 0.001, Fig. 3 ). Similarly, pretreatment with phloretin (300 μM, n = 11), a blocker of swelling-activated Cl⁻ channels, to both surfaces of the tissues inhibited the I_sc increase on hypotonic challenge. The inhibition was significant (P < 0.005), with a mean inhibition of 40% ± 10% (Fig. 3) . 

In addition, pretreatment with flufenamic acid (100 μM), a blocker of Cl⁻ and nonselective cation channels, significantly reduced the hypotonically-triggered I_sc stimulation. Bilateral addition of flufenamic acid for 15 minutes before the hypotonic treatment inhibited the subsequent I_sc increase by 56% ± 7% (n = 11, Fig. 3 ). These results suggest that the increase in I_sc with hypotonic treatment was caused primarily by the transcellular swelling-activated Cl⁻ current directed toward the aqueous humor. 

To confirm that the effect was mediated by Cl⁻ current, we studied the response of hypotonic-triggered I_sc stimulation when the Cl⁻ concentration was low in the bathing solution. Bilateral cyclamate substitution for ∼90% of the bath Cl⁻ inhibited both baseline isotonic I_sc and the effects of bilateral hypotonicity. The partial Cl⁻ replacement reduced I_sc in isosmotic solution from 20.7 ± 1.4 to 2.0 ± 0.6 μA · cm⁻², an inhibition of 90% ± 4% (n = 17, P < 0.001). Subsequently, bilateral hypotonicity increased the absolute value of the I_sc by only 6.1 ± 1.1 μA · cm⁻² (n = 8). Analyzed as for other results, the block was highly significant (P < 0.001) and corresponded to a mean inhibition of 55% ± 7% (Fig. 3) . This result was similar to the effect of NPPB pretreatment under normal Cl⁻ concentration. Addition of bilateral NPPB under low Cl⁻ conditions did not further reduce either the I_sc (P > 0.05, n = 3) or the hypotonically-triggered I_sc stimulation (P > 0.05). In the combined presence of cyclamate substitution for Cl⁻ and NPPB, the I_sc increase was reduced to 5.9 ± 0.6 μA · cm⁻², an inhibition of 62% ± 3%, which was not significantly different from either NPPB or low Cl⁻ alone. Similarly, the combined effect of Cl⁻ substitution and flufenamic acid produced an I_sc increase of 3.7 ± 0.9 μA · cm⁻², accounting for an inhibition of 70% ± 7% (P < 0.001, n = 6; Fig. 3 ). Again, this effect was not different from Cl⁻ replacement alone. These results indicated that stimulation of I_sc produced by bilateral hypotonicity could be inhibited either with the Cl⁻-channel blockers or with Cl⁻ replacement, confirming that swelling-activated Cl⁻ channels contribute significantly to Cl⁻ secretion across the native ciliary epithelium. 

Transepithelial Current Measurements: Sidedness of Hypotonic Effects

As illustrated by Figure 3 , bilateral NPPB inhibited the increase in I_sc triggered by bilateral hypotonicity, suggesting that the effect was primarily caused by the inhibition of NPE-cell Cl⁻ channels at the basolateral membrane. However, swelling has been reported to activate NPPB-inhibitable Cl⁻ channels of isolated PE and NPE cells (Fig. 1) . ²⁰ To test whether PE cells played a role in response to bilateral hypotonic challenge, NPPB was added in a separate series of experiments solely to the stromal surface before and during lowering osmolality of both solutions. Pretreatment of the stromal NPPB for 5 to 15 minutes had no effect on baseline I_sc (n = 23, P > 0.05). In the presence of stromal NPPB, bilateral hypotonicity produced no significant change of hypotonically-triggered I_sc stimulation compared with controls (n = 23, P > 0.05). In contrast, addition of NPPB to the aqueous side significantly inhibited the I_sc increase triggered by bilateral hypotonicity (n = 16, P < 0.05), suggesting that the effect of NPPB can be explained by the inhibition of Cl⁻ channels at the basolateral membrane of NPE cells. 

Cl⁻-Independent Component of Hypotonically-Stimulated Current

We wondered whether the residual increase in I_sc of ∼5 μA · cm⁻² observed even with bilateral NPPB or cyclamate substitution for bath Cl⁻, may have partly reflected paracellular ion movement. Charge transfer across tight junctions of other preparations has been manifest as streaming and/or pseudostreaming potentials (see the Discussion section). If so, unilateral hypotonicity should produce opposite effects when applied separately at the stromal and aqueous surfaces. Hypotonicity applied solely to the aqueous surface is particularly relevant because of the much more direct access of water and solutes from the aqueous than from the stromal surface. Even when hypotonicity is applied to both reservoirs simultaneously, water must traverse the thick stromal tissue to reach the basolateral membranes of the PE cells. 

As shown in Figure 4A , aqueous hypotonicity produced a much more sustained increase in I_sc than did bilateral hypotonicity. The peak increase in I_sc was substantially larger than that produced by bilateral hypotonicity. The mean enhancement was 36% ± 15% (n = 13, P < 0.05). Also, in contrast to the relatively large block of bilaterally triggered changes by NPPB (∼60%, Fig. 3 ), bilateral NPPB had no significant inhibitory effect on aqueous hypotonicity (n = 6, P > 0.05). Stromal hypotonicity exerted an opposite effect, reducing I_sc by 85% ± 7% (n = 7, P < 0.001), in contrast to the stimulatory effects of either bilateral or solely aqueous hypotonicity (Fig. 4B) . The results are consistent with the notion that at least part of the Cl⁻-insensitive fraction of the hypotonically-stimulated current may have reflected net ionic movement through the tight junctions and paracellular pathway (see the Discussion section). 

Cell Volume Measurements

We first verified that the calcein-fluorescence-quenching technique was applicable to the freshly harvested bovine NPE cells. As illustrated in Figure 5A , cells superfused relatively briefly (∼5 minutes) with either hypotonic or hypertonic solutions displayed reversible changes in calcein fluorescence inversely dependent on the osmolality of the solution. We observed a slightly convex-upward dependence of percentage quenching on the relative cell volume calculated as the inverse relative osmotic pressure (π_o/π), where π_o is the isotonic osmolality. The relationship between percentage quenching and relative volume was similar under control conditions and when perfused with a pharmacologic agent (Fig. 5A) , suggesting that the recently described method of calcein-fluorescence quenching is applicable to the NPE cells. 

Exposing the native bovine NPE cells to hypotonic solution produced an initial cell swelling, followed by a slow regulatory volume decrease (RVD) toward the original volume. The initial osmotic cell swelling usually peaked after ∼5 minutes in hypotonic solution, after which cell volume slowly declined. Figures 5B 5C 5Dsummarize cell volume recovery after exposure to hypotonic solution under different experimental conditions. The spontaneous RVD observed in native NPE cells was significantly inhibited by pretreatment (5 minutes) with either 100 μM NPPB (Fig. 5B , n = 4) or 100 μM flufenamic acid (Fig. 5C , n = 4) or by preincubation in Cl⁻-free solution (Fig. 5D , n = 4), supporting the functional importance of Cl⁻ release in mediating the RVD after hypotonic challenge. The duration of the control RVD was ∼15 minutes, similar to that of the increase in I_sc produced by bilateral hypotonicity (Fig. 2B) . 

It was noted that the calcein-fluorescence quenching technique enabled us to measure percentage changes, but not the absolute values of those changes. To reduce the risk of dye loss into the bath, we did not study the sequential effect of Cl⁻-free on cell volume under isotonic and hypotonic conditions, which would have significantly prolonged the experiment. As the volume change on hypotonicity was normalized to the isotonic conditions, the precise shrinkage produced by the preincubation in Cl⁻-substituted solution was not known. However, we have previously measured the absolute cell volumes of human NPE cells in continuous culture using electronic cell sizing. ²¹ In that study, the preincubations in external Cl⁻-free solutions reduced the cell volume by 40% to 50% (Civan MM, Peterson-Yantorno K, Coca-Prados M, Yatorno RE, unpublished observations, 1990) and subsequently inhibited the RVD. 

Discussion

The major findings of the present study are that (1) hypotonic challenge applied at both surfaces of the intact bovine ciliary epithelium triggers an increase in I_sc; (2) the increase can be inhibited by Cl⁻ channel blockers and by Cl⁻ replacement; (3) the volume of native bovine NPE cells can be monitored by a recently described calcein-fluorescence quenching technique, and the measured RVD is sensitive to Cl⁻ channel inhibitors; and (4) the time courses of the increased I_sc triggered by bilateral hypotonicity and of the RVD of isolated NPE cells are similar. 

Hypotonic swelling triggers I_Cl,swell ^{16 17 18 19 20 21} and RVD ^{8 12 21} in isolated NPE cells. Hypotonic challenge is widely used to probe the properties of Cl⁻ channels underlying aqueous humor formation but, to our knowledge, whether swelling-activated Cl⁻ channels are actually involved in ciliary epithelial secretion has never been established. Of particular concern is the distribution of swelling-activated Cl⁻ channels within the ciliary epithelium. Depending on the relative number of these channels in PE and NPE cells and on their orientation in the NPE cells (facing the aqueous or stromal surface), swelling-activation may increase, decrease or have no effect on net Cl⁻ secretion (Fig. 1) . We have addressed this concern by monitoring hypotonically-activated I_sc across the intact bovine ciliary epithelium, an approach followed in studies of several other epithelia. ^{32 33 34 35} Bilateral hypotonicity produced an increase in I_sc, which was inhibited by the Cl⁻-channel blocker NPPB. NPPB has been shown to block K⁺ and nonselective cation channels. ^{36 37} However, K⁺-channel inhibition on the stromal and aqueous surfaces should have inhibited and enhanced I_sc, respectively, opposite to our observation. Also, I_sc was inhibited by the Cl⁻-channel blocker phloretin, which has not been reported to block nonselective cation channels. In addition, bilateral application of flufenamic acid, which is a blocker of both Cl⁻ and nonselective cation channels, produced a similar inhibition of I_sc, as with NPPB alone, suggesting that its effect was primarily mediated by the inhibition of Cl⁻ channels. Furthermore, replacement of the bulk fraction of extracellular Cl⁻ by cyclamate inhibited the hypotonically-triggered increase in I_sc, and both NPPB and flufenamic acid had no further effect on I_sc under low extracellular Cl⁻ concentration. We conclude that hypotonicity applied at both surfaces of the intact bovine ciliary epithelium elicits a net transepithelial I_Cl,swell in the direction that may favor aqueous humor secretion. The implications of this conclusion are that the number, conductance, and/or open-probability of swelling-activated Cl⁻ channels at the aqueous surface of the NPE cells must be greater than those of the NPE and PE cells facing the stromal surface. 

Consistent with our previous findings, ⁵ baseline I_sc reflected primarily the Cl⁻ current because ∼90% of the current was blocked by bath Cl⁻ substitution and by NPPB. Despite this, hypotonic treatment may have elicited both transcellular and paracellular solute movement, because only ∼60% to 70% of the current was sensitive to either Cl⁻ channel blockers or Cl⁻ replacement, or both. The effect was not explained by the stimulation of nonselective cation channels, as flufenamic acid produced no further inhibition of I_sc increase, either at normal or low Cl⁻ concentrations. The basis of the Cl⁻-insensitive increase in I_sc is not entirely clear. However, we suggest that the change in I_sc may reflect paracellular solute movement expressed as streaming and/or pseudostreaming potentials. Streaming potentials arise from net ion flow swept along by net water flow. Pseudostreaming potentials reflect ionic asymmetries across the tight junction established by net water flow. This speculation is supported by the observation that unilateral hypotonicity triggered opposite responses when applied separately to the stromal or aqueous surface of the tissue. Under those circumstances, where the streaming and/or pseudostreaming potentials were enhanced by maintaining a constant asymmetry across the tight junctions, the magnitude of the I_sc response was larger than that expected from bilateral hypotonic challenge. Correspondingly, the change in I_sc produced by unilateral aqueous or stromal hypotonicity was less sensitive to Cl⁻ channel blockers. The reduced contribution of Cl⁻-sensitive currents to the total stimulation produced by unilateral hypotonicity may reflect two factors: Paracellular currents are thought to be enhanced by clamping the osmotic gradient constant, and the NPE cell swelling produced by unilateral, rather than bilateral, hypotonicity is likely to be less, so that the Cl⁻-sensitive swelling-activated transcellular Cl⁻ currents are likely to contribute less to the total observed increase in I_sc. 

The RVD elicited by hypotonic swelling has long been used to study Cl⁻ channel activity in intact NPE cells. ^{14 38} Rather than subject freshly harvested cells to separating procedures that might adversely affect the cells, we tested whether calcein-fluorescence quenching could be applied to monitor cell volume of NPE cells directly identified on coverslips. Anisosmotic changes in solution produced reversible changes in fluorescence quenching. The changes were directly dependent on the magnitudes of the hypertonic and hypotonic changes, reflecting corresponding decreases and increases of cell volume, respectively. It was noted that a small quantity of fluorescence was lost after the swelling, but the decrease in fluorescence did not simply reflect swelling-activated loss of dye because preincubation in low-Cl⁻ solution or blocking Cl⁻ channels with NPPB (100 μM) or both Cl⁻ channels and nonselective cation channels with flufenamic acid (100 μM) markedly inhibited the fluorescence-monitored RVD. The similar time courses of the RVD and the transient increase in I_sc produced by bilateral hypotonicity support the idea that both phenomena reflect swelling-activated Cl⁻ release by the NPE cells. 

In conclusion, this study establishes that the swelling-activated Cl⁻ channels displayed at the cellular level are present in the intact bovine ciliary epithelium and that these channels are oriented in such a way as to subserve aqueous humor formation. These observations support those made in previous studies that were focused on NPE-cell swelling-activated Cl⁻ channels that provide an approach for their study in the intact ciliary epithelium. The results also validate calcein-fluorescence quenching for measuring volume of NPE cells. The common pharmacologic profile, Cl⁻-dependence and time course of hypotonicity-triggered I_sc increase in intact tissue and RVD in dissociated NPE cells suggest that the stimulation of I_Cl,swell facilitates Cl⁻ release by the NPE cells into the posterior chamber, enhancing aqueous humor secretion. In the present study, we imposed large osmotic challenges to enhance the signal-to-noise ratio of the measurements, but in the course of previous studies, ³⁰ we observed higher baseline NPE-cell Cl⁻ currents when the bath osmolality was reduced by only 5 to 10 mOsM. The importance of cell volume, per se, as a major physiologic activator of these channels is unclear but, together with other modulators of NPE-cell Cl⁻ channels, ⁴ is likely to contribute to regulation of aqueous humor inflow. 

 Supported in part by research Grant EY08343 and core Grant EY01583 from the National Institutes of Health.

 Submitted for publication July 1, 2005; revised November 29, 2005, and January 6 and 30, 2006; accepted April 13, 2006.

 Disclosure: C.W. Do, None; K. Peterson-Yantorno, None; M.M. Civan, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

A simplified model of ciliary epithelial secretion showing the potential role of swelling-activated Cl⁻ channels (arrows with dotted lines). As noted, the direction of Cl⁻ transport on hypotonic stimulation is dependent on location, number and activities of swelling-activated Cl⁻ channels.

Figure 2.

 

Effects of bilateral hypotonicity on electrical parameters in native bovine ciliary epithelium. (A) Measurement of transepithelial PD. Constant-current pulses (3 seconds) of 10 μA were applied to the preparation every 5 minutes, and the deflections (ΔPD) were recorded as an index of R. I_sc was calculated from the measured PD and R. The orientation was aqueous surface negative to stromal surface. (B) The calculated I_sc from the same preparation in (A).

Figure 3.

 

Mean inhibition by Cl⁻ channel blockers and Cl⁻ replacement of the increase in I_sc triggered by bilateral hypotonicity. Inhibitions are expressed as percentage of inhibition compared with the control. **P < 0.01 compared with the control; NS, not statistically significant. NPPB: NPPB_(bilateral) (100 μM, n = 12); Flu: Flufenamic acid_(bilateral) (100 μM, n = 11); Phloretin: Phloretin_(bilateral) (300 μM, n = 11); Low Cl⁻: 12 mM Cl⁻ (n = 8); Low Cl⁻ + NPPB: 12 mM Cl⁻ + NPPB_(bilateral) (100 μM, n = 3); Low Cl⁻ + Flu: 12 mM Cl⁻ + Flufenamic acid_(bilateral) (100 μM, n = 6).

Figure 4.

 

The effect of unilateral hypotonicity on I_sc. (A) Measurement of I_sc on applying aqueous hypotonicity in the absence of NPPB pretreatment. (B) A summary of the changes in I_sc in response to unilateral hypotonicity, either in the presence or absence of bilateral NPPB. *P < 0.05 and **P < 0.01 compared with bilateral hypotonicity. Hypo_(both): control (n = 33); Hypo_(aq): aqueous hypotonicity (n = 13); Hypo_(aq) + NPPB: aqueous hypotonicity + NPPB (100 μM, n = 6); Hypo_(st): stromal hypotonicity (n = 7); Hypo_(st) + NPPB: stromal hypotonicity + NPPB (100 μM, n = 4).

Figure 5.

 

Responses of total calcein fluorescence to anisosmotic changes in volume. (A) Changes in fluorescence (ΔF) as a function of brief changes in relative cell size calculated as the inverse relative osmotic pressure (π_o/π) under control conditions (n = 9) and in the presence of 100 μM NPPB (n = 6). (B–D) Regulatory volume decrease (RVD) after hypotonic challenge under different experimental conditions. F_o and π_o symbolize the fluorescence and osmotic pressure under baseline isotonic conditions, respectively. To facilitate comparison, the same control data are presented for NPPB (B), fulfenamic acid (C), and Cl⁻ replacement (D).