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(Investigative Ophthalmology and Visual Science. 2005;46:948-955.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1004
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Inhibition of Swelling-Activated Cl– Currents by Functional Anti-ClC-3 Antibody in Native Bovine Non-Pigmented Ciliary Epithelial Cells

Chi Wai Do,1 Wennan Lu,2 Claire H. Mitchell,1 and Mortimer M. Civan1,3

1From the Departments of Physiology, 2Ophthalmology, and 3Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the potential role of ClC-3, a PKC-inhibitable Cl– channel, in mediating the swelling-activated Cl– current (ICl,swell) of native bovine nonpigmented ciliary epithelial (NPE) cells.

METHODS. Native bovine NPE cells were freshly harvested by enzymatic digestion. Whole-cell currents were recorded by patch–clamp measurements either in the presence or absence of a functional anti-ClC-3 antibody.

RESULTS. Baseline whole-cell currents were small under isotonic conditions. Hypotonic cell swelling stimulated outwardly rectifying ICl,swell, which was reversibly inhibited by the Cl– channel blockers, phloretin (300 µM) or 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 100 µM). Intracellular dialysis with anti-ClC-3 C670-687 antibody did not affect baseline currents, but significantly delayed and inhibited hypotonic stimulation of ICl,swell. Preabsorption of the antibody with its antigen prevented the inhibition of ICl,swell by antibody. In addition, intracellular dialysis with control Ex133-148 antibody did not affect the ICl,swell. Moreover, activation of PKC by pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited the initial stimulation of ICl,swell, but had no effects on the steady state currents.

CONCLUSIONS. The results suggest that endogenous ClC-3 is involved in mediating ICl,swell of native bovine NPE cells. The delayed stimulation of ICl,swell by PDBu may reflect upregulation of swelling-activated Cl– channels of different subtypes, especially when the function of ClC-3 is blocked. This information will be useful in understanding the mechanisms controlling aqueous humor formation and thereby intraocular pressure.

Intraocular pressure (IOP) is a function of the rate of aqueous humor inflow across the ciliary epithelium and of the effective resistance to its outflow from the anterior chamber. Aqueous humor is secreted by the ciliary epithelium, comprising a pigmented ciliary epithelial (PE) cell layer facing the ciliary stroma and a nonpigmented ciliary epithelial (NPE) cell layer in contact with the aqueous humor. Figure 1 presents a minimalist model of aqueous humor inflow,1 2 3 4 5 neglecting the complexities of bidirectionality4 and regional variation6 7 of net secretion. NaCl is taken up by electroneutral transporters of the PE cells, crosses gap junctions to the NPE cells, and is released largely through Na+,K+-activated ATPase and Cl– channels of the NPE cells. Cl– is the major anion of the aqueous humor, and it has been suggested that Cl– efflux by the NPE cells probably limits the rate of inflow.4 This reasoning has been supported by the observations that addition of the Cl– channel blocker 5-nitro-2-(phenylpropylamino)-benzoate (NPPB) to the aqueous surface inhibits net Cl– secretion3 and aqueous humor formation8 in vitro.



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  		FIGURE 1. Consensus model of aqueous humor formation. NaCl is taken up by electroneutral transporters of the PE cells, namely Na+-K+-2Cl– cotransporters and paired Cl–/HCO3– and Na+/H+ exchangers: NaCl then crosses gap junctions to the NPE cells, and is released largely through Na+,K+-activated ATPase and Cl– channels of the NPE cells. CA, carbonic anhydrase.

 
Despite the importance of the NPE Cl– channels in aqueous humor formation, their molecular identities are unknown. The identification of molecular candidates is both scientifically and clinically important in designing selective inhibitors that directly block channel activity or interfere with either their gating or trafficking to the plasma membrane. It has been shown that hypotonic cell swelling activates Cl– channels of native and cultured NPE cells of different species.9 10 11 12 As for many other cells,13 14 15 it remains controversial whether ClC-3 mediates swelling-activated Cl– current (ICl,swell) of NPE cells. Several observations suggest that ClC-3 may encode ICl,swell in NPE cells: (1) Activation of PKC inhibits the ICl,swell in NPE cells, characteristics of ClC-3-associated currents16 17 18 ; (2) NPE cells express ClC-3 mRNA and protein19 ; and (3) antisense oligonucleotides downregulate ClC-3 message, ClC-3 protein expression, and ICl,swell of NPE cells.20 However, the precise role of ClC-3 in NPE cells remains unclear, since ClC-3 antisense maximally inhibits the ICl,swell by only 60%,20 and ClC-3 antisense treatment may interfere with trafficking of other Cl– channels to the plasma membrane, since the great bulk of ClC-3 protein is thought to be perinuclear rather than membrane-bound.

In the present work, we used a recently developed functional anti-ClC-3 C670-687 antibody (Ab)21 to inhibit the endogenous function of ClC-3. This Ab has been demonstrated to block the ICl,swell in freshly harvested canine pulmonary artery smooth muscle cells (PASMCs),21 guinea pig cardiac myocytes,21 and human gastric epithelial cells (AGS).22 This C670-687 Ab clearly recognizes ClC-3, because it detects an immunoblot band at ~90 kDa in brains from homozygous Clcn3+/+ mice but not from Clcn3–/– knockouts.21 This Ab has been considered more specific for ClC-3 than a commercially available Ab directed against residues 592-661 of the carboxyl terminus of ClC-3 (Alomone Laboratories, C592-661).21 22 This assessment is based on the relative absence of additional immunoreactive bands labeled with the new C670-687 Ab in Western blot analysis derived from the cell lines HeLa22 and AGS22 and from PASMCs21 and cardiac tissue.21


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Preparation of Bovine NPE Cells
The procedure for cell isolation and preparation has been previously described.23 Briefly, fresh bovine eyes were obtained from a local abattoir. After removing the cornea and iris, the tips of the ciliary processes were excised and washed with Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen-Gibco, Grand Island, NY). Dissociated ciliary epithelial cells were obtained by incubating with 0.25% trypsin at 37°C for 30 minutes followed by trituration. Thereafter, cells were washed twice with DPBS before seeding onto the coverslips (Fisher Scientific, Pittsburgh, PA). Cells were incubated in medium 199 containing 10% fetal bovine serum and 0.1% gentamicin (Invitrogen-Gibco) at 37°C in 5% CO2 for at least 3 hours, but not >36 hours, before the patch–clamp measurements.

Whole-Cell Patch–Clamp Measurements
Micropipettes for whole-cell configurations with resistance of 3 to 6 M{Omega} were prepared (Corning no. 7052 glass; World Precision Instruments, Sarasota, FL) using a micropipette puller (Flaming/Brown, P-97; Sutter Instrument Co., San Raphael, CA). Micropipettes were then coated (Sylgard; World Precision Instruments) and fire-polished with a microforge (MF-830; Narishige, Tokyo, Japan). Recordings were conducted in a perfusion chamber connected to a Ag/AgCl pellet by a 3 M KCl agar bridge at room temperature (22–24°C). Whole-cell currents were measured with patch clamps (1-B or 1-D patch clamps; Axon Instruments, Foster City, CA) coupled to an external Bessel filter (Model 990C; Frequency Devices, Haverhill, MA). The measurements of whole-cell currents were obtained at 2 kHz and filtered at 500 Hz. The voltage pulses were stepped at 0.5 Hz from a holding potential (Vh) of –40 mV to 300-ms test pulses ranging from –100 to +80 mV in 20-mV increments. Upward current deflections indicated outward currents, and vice versa. Data were acquired with a digital interface (Digidata with Clampex 8.2 software; Axon Instruments), and the results were analyzed with the software. The junction potential between the micropipette filling solution and the bath was calculated with a software program based on the Henderson equation (Clampex 8.2; Axon Instruments).24

Western Blot Analysis
Native bovine ciliary epithelial cells were prepared as mentioned earlier. They were washed twice with cold DPBS and lysed in RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, protease inhibitor cocktail (Complete; Roche Diagnostics, Germany), 1% Triton X-100, 0.1% SDS, and 10% glycerol. The samples were sonicated and cleared by centrifugation (10,000g) for 30 minutes at 4°C. The protein concentrations were determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL). The homogenate containing 20 µg of protein was separated using conventional SDS-PAGE, transferred to the PVDF membrane. Nonspecific binding was blocked with 5% nonfat dried milk for 1 hour at room temperature. Blots were then incubated with either anti-ClC-3 C670-687 Ab alone (1:500) or antigen-preabsorbed C670-687 Ab overnight at 4°C. This was followed by incubation with anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilution; Amersham Biosciences Corp., Arlington Heights, IL) at room temperature for 1 hour and developed by chemiluminescence detection (ECL detection system; Amersham Biosciences Corp.). Fresh bovine brain tissue was used as a positive control.

Solutions and Pharmacological Agents
For whole-cell patch–clamp measurements, the micropipette solution contained (in mM): 24.2 NaCl, 110 aspartic acid, 120 N-methyl-D-glucamine base, 0.38 CaCl2, 0.8 NaHEPES, 11.2 HEPES, 1.0 EGTA, 1.0 MgATP, and 0.01 GTP (280 mOsmol/kg H2O, pH 7.2). The baseline isotonic bath contained (in mM): 110 NaCl, 6 HEPES, 6 NaHEPES, 1.8 CaCl2, 1.2 MgCl2, 5 glucose, 67 mannitol (310 mOsmol/kg H2O, pH 7.4). The hypotonic solution was prepared by removing the mannitol from the solution (240 mOsmol/kg H2O, pH 7.4).

All chemicals were reagent grade. Phorbol 12,13-dibutyrate (PDBu), phloretin, and 5-nitro-2-(phenylpropylamino)-benzoate (NPPB) were obtained from Sigma-Aldrich (St. Louis, MO). The polyclonal anti-ClC-3 C670-687 Ab and control Ab extracellular epitope (Ex133-148) were gracious gifts of Joseph R. Hume and his laboratory (Department of Pharmacology, University of Nevada School of Medicine, Reno, NV). The C670-687 Ab was produced from the peptide epitope that corresponds to residues 670-687 at the carboxyl terminus of mouse ClC-3 (short form).21 For the patch–clamp experiments, the anti-ClC-3 Ab or antigen-preabsorbed Ab was added to the micropipette solution. For antigen-preabsorbed Ab, the Ab was mixed with corresponding antigen in a ratio of 1:20 and stored at 4°C overnight before the experiments. The final concentration of the C670-687 Ab in the micropipette was adjusted to 10 µg/mL.21 25 The osmolarity of the solution was not significantly affected by the inclusion of either Ab or antigen-preabsorbed Ab.

Statistics
The data were expressed as means ± SEM, where n was the number of experiments. The statistical differences were evaluated using Student’s paired or unpaired t-test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Activation of ICl,swell of Native Bovine NPE Cells
With 25 mM NaCl in the micropipette, the baseline whole-cell currents were small (–0.7 ± 0.1 pA/pF at –80 mV and 1.9 ± 0.3 pA/pF at +80 mV, n = 27) under isotonic conditions. Hypotonic solution triggered a gradual stimulation of swelling-activated currents in native bovine NPE cells (Fig. 2a) to mean steady state peaks of –20.2 ± 2.6 pA/pF at –80 mV and 84.8 ± 12.6 pA/pF at +80 mV (n = 20). The swelling-activated currents displayed outward rectification with little inactivation at large depolarized potentials (+80 mV; Fig. 2b ). In most preparations, stimulation of currents started ~2 to 3 minutes after applying hypotonicity and reached a steady state in ~15 minutes. The measured reversal potential in the steady state was –27 ± 1 mV (n = 20). Taking the junction potential into consideration, the corrected reversal potential was estimated to be –33.1 mV, which was close to the Nernst potential for Cl– (–38.8 mV). This difference could be ascribed to a small relative permeability of aspartate (~0.06) with respect to Cl– through the swelling-activated channels.



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  		FIGURE 2. A representative experiment showing the stimulation of ICl,swell of native bovine NPE cells. The Cl– concentrations in the micropipette and in the bath were 25 and 122 mM, respectively. Voltage pulses were stepped from –100 to +80 mV in 20-mV increments at a holding potential (Vh) of –40 mV. (a) Time course of the stimulation of ICl,swell. The activation of ICl,swell began ~2 minutes after addition of hypotonic solution. (b) Current traces illustrating the outwardly rectifying ICl,swell, with little time-dependent inactivation at depolarized pulses (+80 mV). (c) Current-voltage relationship illustrating the stimulation triggered by hypotonic solution with a corrected reversal potential of approximately –33 mV. Iso, isotonic solution; Hypo, hypotonic solution.

 
Cl–-channel blockers such as phloretin and NPPB were shown to inhibit swelling-activated currents significantly (Figs. 2 3) , further suggesting that the currents were primarily carried by Cl– (ICl,swell). Phloretin has been shown to be a relatively selective blocker of ICl,swell.26 At 300 µM, it preferentially blocks the ICl,swell, and not Ca2+-dependent Cl– currents in human colonic T84 cells.27 In the present study, after 15 minutes of hypotonic cell swelling, addition of 300 µM phloretin to the bath caused a significant inhibition of ICl,swell in native NPE cells; the inward and outward currents were inhibited by 75% ± 6% and 84% ± 4%, respectively (n = 13). Similarly, NPPB (100 µM) produced an inhibition of both inward and outward whole-cell currents by 63% ± 8% and 79% ± 6%, respectively (n = 4). In both cases, the effects were rapid and reversible. As demonstrated in other epithelial cells,28 29 phloretin could activate Ca2+-dependent K+ channels; this possibility was precluded in our experiments by using K+-free solutions in both micropipette and bath solutions.



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  		FIGURE 3. Inhibition of ICl,swell by Cl–-channel blockers. After exposure to hypotonic solution for 15 minutes, either 300 µM phloretin (n = 13) or 100 µM NPPB (n = 4) was added to the extracellular bath. Mean whole-cell currents were measured at ±80 mV. The results are expressed as means ± SEM, as indicated by the vertical bars. **P < 0.01.

 
Inhibition of ICl,swell by Intracellular Dialysis with Anti-ClC-3 C670-687 Ab
Intracellular dialysis of native bovine NPE cells with 10 µg/mL anti-ClC-3 C670-687 Ab for 10 minutes did not affect the baseline whole-cell currents under isotonic conditions (–0.5 ± 0.1 pA/pF at –80 mV and 1.6 ± 0.4 pA/pF at +80 mV, n = 8). There was no significant difference in baseline inward and outward currents between control cells and cells dialyzed with anti-ClC-3 Ab (P > 0.05). However, the inclusion of C670-687 Ab significantly delayed the activation of ICl,swell and diminished the amplitude of ICl,swell during hypotonic cell swelling. Figure 4 compares the activation of ICl,swell in the presence of either anti-ClC-3 Ab or antigen-preabsorbed Ab during hypotonic treatment. The activation of ICl,swell after hypotonic challenge was significantly delayed by ~6 to 8 minutes in the presence of anti-ClC-3 Ab. At the steady state, the whole-cell currents with the inclusion of anti-ClC-3 Ab were inhibited by 87% at –80 mV and 88% at +80 mV compared with control cells (n = 7, P < 0.01). Although the amplitude of ICl,swell was significantly reduced in preparations with anti-ClC-3 Ab, the residual whole-cell currents activated by osmotic cell swelling demonstrated similar biophysical characteristics to those of control cells (Fig. 4b 4c 4d) . The currents displayed similar degrees of outward rectification with little time-dependent inactivation at +80 mV. In addition, the reversal potential was not different from those of control preparations (–27 ± 2 mV, n = 7, P = 0.82). Similarly, ICl,swell activated either in the presence or absence of anti-ClC-3 Ab was phloretin-sensitive; addition of 300 µM phloretin to the bath produced a similar degree of current inhibition. For those cells exposed to anti-ClC-3 Ab, phloretin reduced the inward and outward currents by 61% ± 8% (–3.0 ± 0.9 to –0.7 ± 0.2 pA/pF) and 71% ± 8% (14.1 ± 4.9 to 1.8 ± 0 pA/pF), respectively (n = 12).



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  		FIGURE 4. Intracellular dialysis of native bovine NPE cells with anti-ClC-3 C670-687 Ab. (a) Measurements of inward and outward currents at ±80 mV in the presence of either anti-ClC-3 Ab or antigen-preabsorbed Ab. (b) Current traces illustrating the inhibitory effects on ICl,swell by anti-ClC-3 Ab. (c, d) I-V relationships showing the outwardly rectifying Cl– currents with similar reversal potentials in cells dialyzed with either (c) anti-ClC-3 C670-687 Ab or (d) antigen-preabsorbed Ab.

 
To determine whether the inhibition of ICl,swell was specific to ClC-3, three different strategies were used. First, we monitored the activation of ICl,swell with the inclusion in the micropipette of anti-ClC-3 C670-687 Ab preabsorbed with the corresponding antigen. As shown in Figures 4 and 5 , the inhibition of ICl,swell by Ab was significantly prevented in cells exposed to antigen-preabsorbed Ab. Moreover, the addition of antigen alone to the micropipette solution had no significant effect on ICl,swell at all tested potentials (P > 0.05, n = 8). In both cases, the reversal potentials were not different from control cells (P > 0.05). Second, we intracellularly dialyzed 1.6 µg/mL control Ex133-148 Ab. This Ab was raised against an extracellular epitope of ClC-3 and did not significantly affect ICl,swell (Fig. 5) , supporting a specific action of C670-687 Ab on ClC-3. Third, the anti-ClC-3 C670-687 Ab detected a prominent band of ~90 kDa in Western blot analysis from native bovine ciliary epithelial cells and brain (Fig. 6) . This band conformed to the expected position of ClC-3 protein. In contrast, these bands were not detected by preabsorbed Ab, confirming a specific interaction of C670-687 Ab and ClC-3.



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  		FIGURE 5. Effects of intracellular dialysis with anti-ClC-3 C670-687 Ab on ICl,swell in native bovine NPE cells. The ICl,swell was measured at –80 and +80 mV in control cells (n = 20–27) and in cells dialyzed with control Ex133-148 Ab (n = 7), anti-ClC-3 C670-687 Ab (n = 7–8), and antigen-preabsorbed ClC-3 C670-687Ab (n = 9–11). The results are expressed as the mean ± SEM, as indicated by the vertical bars. *P < 0.05; **P < 0.01.

 


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  		FIGURE 6. Western blot analysis of ClC-3 protein in bovine ciliary epithelial cells using the specific anti-ClC-3 C670-687 Ab. Brain tissue was used as a positive control. The recognition of ClC-3 protein (~90 kDa) was demonstrated (left) in both bovine ciliary epithelial cells (CE) and brain cells (B). With the antigen-preabsorbed Ab (right), no bands were detected in both cases.

 
Delayed Activation of ICl,swell by Pretreatment with PDBu
ClC-3-associated currents can be inhibited by activation of PKC.30 Similar to the results with anti-ClC-3 Ab, pretreatment of native bovine NPE cells with a commonly used PKC activator, phorbol 12,13-dibutyrate (PDBu), for 1 hour did not affect the baseline whole-cell currents under isotonic conditions (n = 15, P > 0.05). In contrast, pretreatment with 100 nM PDBu significantly delayed the initial activation of ICl,swell by hypotonic cell swelling. At 10 minutes after osmotic swelling, the inward and outward currents were inhibited by 48% (P < 0.05, n = 10) and 37% (P < 0.05, n = 10), respectively (Fig. 7) . However, the initial inhibition of ICl,swell was not maintained under steady state conditions, ~15 minutes after application of hypotonicity. No significant difference of ICl,swell was observed in the steady state between PDBu-pretreated and untreated cells. The inward and outward currents were –19.5 ± 4.5 pA/pF (n = 9, P = 0.88) and 83.9 ± 14.6 pA/pF (n = 9, P = 0.97), respectively. Addition of 300 µM phloretin produced a comparable inhibition of ICl,swell in both directions compared with control cells (Fig. 7) .



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  		FIGURE 7. Delayed activation of ICl,swell in native bovine NPE cells by pretreatment with 100 nM PDBu. NPE cells were pretreated with PDBu for 1 hour before the hypotonic challenge. *P < 0.05; **P < 0.01.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
In the present study, we focused on ICl,swell as a probe of the NPE-cell Cl– channels. Whether the same Cl– channels subserve both ICl,swell and the final physiologic step in aqueous humor formation has not been rigorously established. However, ICl,swell and the A3 adenosine-receptor (AR)-activated Cl– channels of transformed human NPE cells share several biophysical properties, including moderate outward rectification, anionic selectivity, and inhibition by cyclamate.24 It is plausible, albeit as yet unproven, that cell swelling may also be a physiologic trigger for activating NPE-cell Cl– channel activity that thereby enhances the rate of aqueous humor inflow.31 According to the consensus model of Figure 1 , cell swelling is thought to arise from transfer of NaCl and water from the stroma through the PE cells and gap junctions to the NPE cells. However, other stimuli may well play more important roles in triggering activation of NPE cell Cl– channels. For example, knockout of A3ARs has been found to lower IOP in the living mouse,32 at least in part by reducing inflow.24 33 34

The major results of the present study of freshly harvested NPE cells are that a selective functional Ab to ClC-3 can block ICl,swell and that PKC activation by pretreatment with PDBu can delay but not block ICl,swell. The possibility that ClC-3 encodes the ICl,swell of cells generally has been difficult to resolve.13 14 35 As discussed elsewhere,36 at particular issue have been the inability of some experienced laboratories to express ClC-3-associated currents13 and the retention of ICl,swell in cells harvested from ClC-3-knockout mice.37 However, the functional properties of ICl,swell in cells from such transgenic animals have been reported recently to differ from those of wild-type mice, suggesting that other proteins can compensate to support ICl,swell after ClC-3 gene disruption.25

Our results showed that the baseline whole-cell currents were small under isotonic conditions and were unaffected by the anti-ClC-3 C670-687 Ab. Hypotonic cell swelling triggered ICl,swell in native bovine NPE cells, increasing the whole-cell currents by 1 to 2 orders of magnitude. ICl,swell was shown to exhibit moderate outward rectification with little time-dependent current inactivation at depolarizing pulses. It has been suggested that the degree of depolarization-triggered inactivation displayed by Cl– channels in many cells is influenced by free intracellular Mg2+ concentration and other unidentified factors.28 Intracellular dialysis with anti-ClC-3 C670-687 Ab delayed and inhibited the activation of ICl,swell by ~80% to 90%, suggesting a substantial role for ClC-3 in mediating the ICl,swell of the native bovine NPE cells. The carboxyl terminus anti-ClC-3 C670-687 Ab is designed to recognize epitopes of both short and long forms of ClC-3,21 since both forms are identical except for an additional 58 amino acids at the amino terminus of the long form.38 The ICl,swell inhibition was selective since preabsorbing the Ab with corresponding antigen significantly reduced the inhibitory effects of C670-687 Ab on ICl,swell. The lack of inhibition by the antigen-preabsorbed Ab suggests that the inhibitory effect did not result from nonspecific binding. Consistent with this finding, intracellular dialysis with a control Ab produced no significant effects on ICl,swell. In addition, Western blot analysis of bovine ciliary epithelial cells demonstrated the specific recognition of ClC-3 protein, using the C670-687 Ab, which had not been detected when cells were incubated with preabsorbed Ab. Our observations are in qualitative agreement with a previous study of ClC-3 in the same cell type, but using a different strategy.20 In that study, ClC-3 antisense oligonucleotide delayed and inhibited the activation of ICl,swell by a maximum of 60%. Also, the ClC-3 antisense approach was shown to reduce the expression of endogenous ClC-3 predominantly in the nuclear region, making it unclear whether the remaining portion of ICl,swell was mediated either by other swelling-activated channels or by membrane-associated ClC-3 channels. Depending on the turnover rate of endogenous ClC-3 protein, the ClC-3 antisense strategy may underestimate the functional importance of existing membrane-associated ClC-3 channels, possibly accounting for the larger percentage inhibition of ICl,swell noted with the functional Ab approach.

PKC exerts different effects on ICl,swell in different cell preparations. Activating PKC inhibits ICl,swell of some cells,39 40 but exerts the opposite effect in others.41 42 Recent work has also suggested that different subtypes of swelling-activated Cl– channels, with and without sensitivity to PKC, may be expressed in the same cell.25 The inhibition of ClC-3-associated Cl– channels by PKC appears to be mediated by protein dephosphorylation at a PKC consensus site.39 Consistent with the Ab results that ClC-3 mediates NPE-cell ICl,swell, PKC activation delayed the appearance of ICl,swell. After 5 to 10 minutes of hypotonic superfusion, ICl,swell was inhibited by ~40% to 50%. However, the inhibition was not sustained; no reduction in ICl,swell was observed later in the steady state. Our results are consistent with the notion that the activities of other swelling-activated Cl– channels that are not regulated by PKC may have been upregulated when the activity of ClC-3 was blocked.43 44 45 This view is further supported by the incomplete inhibition of ICl,swell by anti-ClC-3 C670-687 Ab and is consistent with the recent findings of compensatory expression of other proteins in response to ClC-3 gene disruption.25 Our result was different from that in cultured rabbit NPE cells in which the pretreatment of PDBu inhibited the ICl,swell measured after 30 minutes of hypotonicity.40 However, this may reflect different rates of compensatory changes in response to the hypotonicity in different cell types.

The delayed activation of ICl,swell with PDBu pretreatment was different from the effects of ClC-3 antisense oligonucleotide20 or anti-ClC-3 Ab in establishing a relatively constant inhibition of ICl,swell in the steady state. The reasons for the differential effects of PDBu and anti-ClC-3 Ab are unclear, but may reflect the possibilities that (1) the activation of PKC by PDBu may be transient and that PDBu can initially activate and then downregulate PKC in some cells46 ; (2) anti-ClC-3 Ab and PDBu may inhibit ICl,swell by different mechanisms, especially since anti-ClC-3 C670-687 Ab binds to the carboxyl terminus, whereas PDBu acts on the amino terminus of ClC-3 channels39 ; (3) ClC-3 has at least two different isoforms that may have distinct functions and sensitivity to PKC activators and inhibitors, leading to differences in regulation of ICl,swell47 48 ; and (4) the anti-ClC-3 Ab may interfere with trafficking or stimulation of different swelling-activated Cl– channels of different subtypes capable of mediating ICl,swell. Further studies are required to test these hypotheses.

In summary, the inhibition of ICl,swell by anti-ClC-3 C670-687 Ab and the dependence of Cl– currents on PKC activity is consistent with the possible importance of endogenous ClC-3 protein in regulating Cl– channel activity of native bovine NPE cells. The differential effects of inhibiting ClC-3 by a selective functional Ab and by blocking PKC activity may reflect different roles of ClC-3 as part of a plasma-membrane Cl– channel and as a regulator in expressing other membrane-associated Cl– channels in NPE cells. Identifying the mechanisms and proteins involved in aqueous secretion may lead to a better understanding of how current anti-glaucoma drugs reduce secretion and could lead to design of new therapeutic approaches to forestall optic nerve and retinal diseases.


   Acknowledgements
 
The authors thank Joseph R. Hume and his colleagues, Gexin Wang and William J. Hatton, for helpful discussions and Mei Kong and Julian Lum (Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA) for invaluable suggestions and assistance.


   Footnotes
 
Supported by research Grant EY08343 and Core Grant EY01583 from the National Eye Institute.

Submitted for publication August 20, 2004; revised October 26 and November 16, 2004; accepted November 20, 2004.

Disclosure: C.W. Do, None; W. Lu, None; C.H. Mitchell, 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.

Corresponding author: Mortimer M. Civan, Department of Physiology, University of Pennsylvania, Richards Building, Philadelphia, PA 19104-6085; civan{at}mail.med.upenn.edu.


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

  1. To CH, Do CW, Zamudio AC, Candia OA. Model of ionic transport for bovine ciliary epithelium: effects of acetazolamide and HCO3–. Am J Physiol. 2001;280:C1521–C1530.
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