Association of intrinsic pICln with volume-activated Clminus current and volume regulation in a native epithelial cell

Lixin Chen, Liwei Wang, and Tim J. C. Jacob

School of Biosciences, Cardiff University, Cardiff CF1 3US, United Kingdom

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We investigated the relationship between pICln, the volume-activated Cl- current, and volume regulation in native bovine nonpigmented ciliary epithelial (NPCE) cells. Immunofluorescence studies demonstrated the presence of pICln protein in the NPCE cells. Exposure to hypotonic solution activated a Cl- current and induced regulatory volume decrease (RVD) in freshly isolated bovine NPCE cells. Three antisense oligonucleotides complementary to human pICln mRNA were used in the experiments. The antisense oligonucleotides were taken up by the cells in a dose-dependent manner. The antisense oligonucleotides, designed to be complementary to the initiation codon region of the human pICln mRNA, "knocked down" the pICln protein immunofluorescence, delayed the activation of volume-activated Cl- current, diminished the value of the current, and reduced the ability of the cells to volume regulate. We conclude that pICln is involved in the activation pathway of the volume-activated Cl- current and RVD following hypotonic swelling.

antisense oligonucleotides; ciliary epithelium; secretion; fluid transport; ion channels; nonpigmented ciliary epithelial cells

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

A VOLUME-ACTIVATED Cl- current has been observed in many cell types (2, 8, 9, 21, 22, 25, 26, 28, 30, 31, 33, 37). This current plays an important role in the regulation of cell volume. It has been reported that four proteins, ClC-2 (15), ClC-3 (10), pICln (32), and P-glycoprotein (13, 37), are associated with this current in some cell types.

The nonpigmented ciliary epithelial (NPCE) cells, one of the two types of cells in the ciliary epithelium, play a critical role in the secretion of the aqueous humor. Volume-activated Cl- channels are considered to be involved in the formation of aqueous humor (20). During secretion, the main anion secreted is Cl- (4, 24), and Cl- channels are rate limiting for aqueous humor secretion (6). It has been demonstrated by patch-clamp studies that the ciliary epithelial cells possess three volume-activated Cl- channels, two of which are found in the nonpigmented cells (44), but the molecular basis for these channels is not clear. Hypotonic shock could activate a Cl- current in these cells (42), and this current was associated with P-glycoprotein (the product of multidrug resistance 1 gene). pICln has been cloned and functionally expressed in NPCE cells (1, 6, 39) and has been suggested to play an important role in the activation of volume-activated Cl- current. Coca-Prados and colleagues (7) showed that the NPCE cells express transcripts for ClC-3 and suggested that ClC-3 was the volume-activated Cl- channel involved in volume regulation. In their model, the volume-sensitive pICln was tethered in the vicinity of the channel through actin binding sites (32) and regulated the activity of the ClC-3 channel (7). Paulmichl et al. (32) proposed that pICln was an anion channel-forming pore on the basis of Xenopus expression studies. The protein possessed a nucleotide-binding site near the putative channel pore, consistent with the inhibition of the pICln-associated current by extracellular nucleotides. Furthermore, mutations to this site rendered the anion current insensitive to nucleotides. Paulmichl and colleagues (16) went on to demonstrate a link between pICln and cell-swelling activated Cl- currents using antisense oligonucleotides and with cell volume regulation by nucleotide inhibition. However, work demonstrating the cytoplasmic location of pICln in oocytes (23) and rat C6 glioma cells (11) cast some doubt on its role as a membrane channel. This led to the postulation of the "anchor-insertion" model of channel activation (35), in which the stimulus of cell swelling causes the translocation of pICln to the membrane and its insertion in an "active" state into the membrane. This explained the jumps in channel activity seen upon cell swelling. In support of this, pICln has been shown to be translocated from the cytoplasm to the membrane during cell swelling (14, 27, 29) and, in a study on red blood cells, to be associated with the membrane (32a). Recently, however, Emma et al. (11) were unable to find any translocation of the cytoplasmic pICln signal to the membrane following cell swelling in rat C6 glioma cells. Does this spell the end of the candidacy of pICln for the volume-activated Cl- current? In this study, we demonstrate the presence of pICln in a native cell, its association with the volume-activated Cl- current, and its involvement with cell volume regulation.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of cells. The NPCE cells were prepared by a method similar to that described previously (19). The tips of the ciliary body were dissected from bovine eyes and dissociated using 0.25% trypsin (Sigma, Poole, Dorset, UK) with 0.02% EDTA in a Ca2+- and Mg2+-free buffer for 30-40 min at 37°C. The tissue was triturated in a solution of culture medium 199 (Sigma) with 10% FCS, spun at 500 g for 5 min, and washed twice. The cells were suspended in medium (medium 199 plus 10% FCS) and plated on 6-mm uncoated glass coverslips, which were then put into 24-well tissue culture plates and incubated overnight (14-18 h) at 37°C to allow the cells to attach and recover from trauma associated with enzymatic digestion.

Oligonucleotides. The four HPLC-purified oligonucleotides were synthesized by Severn Biotech (Milton Keynes, UK). The sense sequence, from base -3 to base +12, was 5'-GCT ATG AGC TTC CTC-3'. The sequences of antisense 1, 5'-CGG CGG CGG GAA ACT TTT GAG GAA GCT CAT-3', and antisense 2, 5'-TTT GAG GAA GCT CAT-3', are complementary to the initiation codon region of the human pICln mRNA (1) starting at the initiation codon. The sequence of antisense 3, 5'-GAG GAA GCT CAT AGC-3', is also complementary to the initiation codon region of the human pICln mRNA, but starting from the third base before the initiation bases (ATG). The first three bases at each end of the three antisense oligonucleotides were phosphorothioated. For measurement of the oligonucleotide uptake by cells, the oligonucleotides were labeled with fluorescein at the 11th and 20th bases in antisense 1, at the 1st base at each end in antisense 2, and at the 5th and the 11th bases in antisense 3.

Oligonucleotide treatment of cells. The cells, attached to coverslips and incubated overnight, were rinsed with serum-free medium 199. Serum-free medium 199 (0.5 ml), with or without oligonucleotide and with or without Lipofectin (GIBCO BRL, Paisley, UK), was added to each well of a 24-well plate, each well of which contained three 6-mm glass coverslips. The cells were then cultured for 24 or 48 h before fluorescence measurements or for 48 h before electrophysiological recordings, immunofluorescence experiments, and volume measurements.

Whole cell recording. Whole cell currents of single NPCE cells were recorded using the patch-clamp technique previously described (19) with a List EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany). Electrodes were pulled from standard wall borosilicate glass capillaries with inner filament (Clark Electromedical Instruments, Pangbourne, Kent, UK) on a two-stage vertical puller (PB-7, Narishige, Tokyo, Japan) and gave a resistance of 5-10 MOmega when filled with the electrode solution. The junction potential was corrected when the electrode entered the bath. Voltage and current signals from the amplifier, together with synchronizing pulses, were digitized using a CED 1401 laboratory interface [Cambridge Electronic Design (CED) Cambridge, UK], with a sampling rate of 1 kHz and recorded on computer disks using a personal computer. The voltage pulse generation and current analysis were performed with the EPC software package (CED).

The standard voltage protocol used to record currents was as follows. The cells were held at the Cl- equilibrium potential (0 mV) and then polarized to -40, +40, 0, -80, and +80 mV, with 200 ms at each potential and 4 s at 0 mV between each step. The cells were continuously cycled through the voltage protocol. All current measurements were made 10 ms after the onset of each voltage pulse.

Solutions. Special solutions were used to record Cl- currents. The pipette solution contained (in mM) 105 N-methyl-D-glucamine chloride, 1.2 MgCl2, 10 HEPES, 1 EGTA, 70 D-mannitol, and 2 ATP. The isotonic bath solution contained (in mM) 105 NaCl, 0.5 MgCl2, 2 CaCl2, 10 HEPES, and 70 D-mannitol. The hypotonic bath solution (23% hypotonic) was obtained by simply omitting the D-mannitol from the solution. The osmolarity in both the pipette solution and in the isotonic solution was adjusted to 300 mosmol/l with sucrose. The pH of the pipette solution and the pH of the bath solution were adjusted to 7.25 and 7.4, respectively, with Tris base.

Fluorescence measurement. The cells were prepared as above. After 24 or 48 h of incubation with or without fluorescein-labeled oligonucleotide and with or without Lipofectin, the cells, attached to glass coverslips, were washed with bath solution twice and then examined with an Odyssey real-time laser confocal microscope (Noran Instruments, Middleton, WI). Fluorescence from control and experimental cells was measured and quantified on the same day using the same excitation beam strengths and computer settings. The focus was adjusted until the peak signal was obtained, the images were acquired, and the gray levels of the images of the nonpigmented cells were measured by using MetaMorph image analysis system (Universal Imaging, West Chester, PA). The fluorescence (gray level) values are expressed in units on an 8-bit scale, in which 0 = black and 255 = white.

Immunofluorescence. Cells from the ciliary epithelium were prepared and treated in exactly the same way as for electrophysiology. The cells, attached to the coverslips, were washed with PBS and fixed in 4% paraformaldehyde (plus 0.12 M sucrose) in PBS. The cell membranes were permeabilized with 0.5% Triton X-100 in PBS and blocked with 10% sheep serum (Sigma). The cells were then incubated in a refrigerator overnight in the presence and absence of the primary antibody, rabbit anti-pICln antibody (a kind gift from Markus Paulmichl's laboratory, University of Innsbruck, Innsbruck, Austria) diluted 1:10 in 1% sheep serum and PBS. Next, they were washed with PBS and incubated for 1 h in the dark with sheep anti-rabbit IgG conjugated to FITC (Sigma), diluted 1:100 in 1% sheep serum and PBS. Finally, the coverslips were washed with PBS, inverted onto Vectashield mounting medium (Vector Laboratories) on glass slides, sealed with nail polish, and examined by confocal microscopy.

Volume measurements. Volume changes of the cells were followed using a light reflection and light scattering technique. The cells were prepared and treated with or without oligonucleotide and Lipofectin in exactly the same way as for the electrophysiological studies. The glass coverslips containing the cells were fixed in a special holder and then placed into a perfused cuvette in a luminescence spectrometer (Perkin Elmer, Beaconsfield, Bucks, UK) at 45° to the incident beam of light. The cells were illuminated with an excitation beam of 345 nm, and reflected light was collected by the detector. The emission wavelength was set to 392 nm to avoid saturating the detector. The cells swelled following exposure to hypotonic solution, the cell swelling caused more scattering and less reflection, and thus the intensity of the light collected decreased.

Statistics. Data are expressed as means ± SE (n is the number of observations) and where appropriate were analyzed using Student's t-test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Uptake of antisense oligonucleotides. To facilitate the uptake of oligonucleotides, the transfection reagent Lipofectin was added to the culture medium together with antisense oligonucleotides labeled with fluorescein. The uptake was monitored by confocal fluorescence microscopy. Almost all cells took up the fluorescein-labeled oligonucleotides (see Fig. 1). The pairs of pictures in Fig. 1 (A and B, C and D) represent the light and confocal images of the cells 48 h after treatment. There was some very weak background fluorescence in the control groups (Fig. 1, A and B), but the fluorescence in the cells was increased greatly in antisense 2 groups (Fig. 1, C and D). The same results were obtained with antisense 1 and antisense 3 (data not shown). Figure 2 shows that the uptake of oligonucleotide by the NPCE cells was dose dependent and that the gray level of fluorescence in the cells after 24 h was not significantly different from that after 48 h. The gray levels were 45.8 ± 7.7 units (24 h; n = 17) and 61.9 ± 5.5 units (48 h; n = 15) in antisense 1 (200 µg/ml; Fig. 2A), 54.9 ± 4.6 units (24 h; n = 20) and 50.4 ± 4.6 units (48 h; n = 32) in antisense 2 (200 µg/ml; Fig. 2B), and 78.2 ± 7.6 units (24 h; n = 22) and 77.3 ± 7.7 units (48 h; n = 18) in antisense 3 (200 µg/ml; Fig. 2C). The data suggest that the cytoplasmic levels of oligonucleotides are directly dependent on the external levels. There appears to be a difference in the kinetics of uptake between the different antisense oligonucleotides. The process of uptake is unclear. Although it is enhanced by the coadministration of cationic lipids (e.g., Lipofectin), uptake may occur by fluid-phase endocytosis (pinocytosis), perhaps mediated by receptor-like recognition, and it may depend on such factors as oligonucleotide chain length and class (38).


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Fig. 1.   Uptake of fluorescently labeled oligonucleotides. Light micrographs (A and C) are of same images as laser scanning confocal microscope images (B, D). Ciliary epithelial cells were incubated in control solution (A and B) or human pICln antisense oligonucleotide (antisense 2, 200 µg/ml) with transfecting agent Lipofectin (20 µg/ml; C and D) for 48 h. Objective magnification, ×20.


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Fig. 2.   Dose-dependent uptake of fluorescently labeled oligonucleotides. Uptake of fluorescence is represented as a gray level (8-bit scale; 0 = black, 255 = white) measured by confocal microscopy. There was no significant difference in fluorescence between control (CTRL; no additives; n = 18) and Lipofectin alone (20 µg/ml; n = 27; data not shown) incubated for 48 h. However, gray level (fluorescence) in nonpigmented ciliary epithelial (NPCE) cells increased in a dose-dependent manner after treatment with 20 µg/ml Lipofectin and with fluorescently labeled antisense 1 (A), antisense 2 (B), and antisense 3 (C) for 24 and 48 h. LA10, LA50, LA200, LA300, and LA400 denote treatment with 10, 50, 200, 300, and 400 µg/ml labeled antisense oligonucleotides, respectively.

Volume-activated Cl- current. The whole cell currents in single NPCE cells were recorded using patch-clamp technique 48 h after treatment with oligonucleotide and/or Lipofectin. Figure 3 illustrates the whole cell current in response to voltage steps of -40, +40, 0, -80, and +80 mV. The cells were first placed in a perfused recording chamber that contained isotonic solution. Under these conditions, whole cell currents in response to voltage steps of -40, +40, 0, -80, and +80 mV were steady and small (Fig. 3A). The isotonic bathing solution was exchanged for hypotonic bathing solution 2 min after establishment of the stable whole cell configuration. Hypotonically induced currents were activated after a period of time that was taken as the latency and reached a peak gradually (Fig. 3B). The currents showed outward rectification and reversed at a voltage that was close to the equilibrium potential for Cl- (Fig. 3C). The bathing solution was then changed back to isotonic solution 7 min after the activation of the Cl- currents, and this led to the gradual reduction of the volume-activated currents to control levels (Fig. 3D). Under these conditions, we have demonstrated that the currents activated are ATP dependent and carried by Cl- (42). During the experiments, the cells were visually monitored under the microscope. The cells appeared swollen and remained so under hypotonic condition until isotonic solution was returned to the bath.


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Fig. 3.   Whole cell voltage-clamp recordings from single NPCE cells. Patch pipette contained a buffer designed for examination of Cl- current (see METHODS). Cells were bathed in isotonic solution (~10 min) before being exposed to a 23% hypotonic solution. This was followed by a wash in isotonic solution while cells were cycled through voltage protocol (-40, +40, 0, -80, and +80 mV; see METHODS). A: current traces obtained in isotonic solution. B: current traces obtained in hypotonic solution. C: current (I)-voltage (V) plots under isotonic condition (Iso) and hypotonic shock (Hypo). D: time dependence of hypotonic experiment. Currents were measured 10 ms after beginning of each voltage pulse.

In the control group (no additives), the latency of the activation of the Cl- currents was 124 ± 29 s (n = 11), after which the currents increased. The peak current elicited by a step of +80 mV (taken at 7 min after latency) was 1,599 ± 116 pA (n = 11).

Lipofectin (20 µg/ml) was used to introduce the oligonucleotides into the cells. There were no significant differences in the latency or the value of currents activated by hypotonic shock between the Lipofectin group and the control group. The latency and the value of the peak current in the Lipofectin group were 126 ± 17 s and 1,431 ± 92 pA (n = 29), respectively, at the +80-mV step.

pICln antisense oligonucleotides inhibited the activation of volume-activated current. The volume-activated Cl- currents were inhibited by incubating the cells with antisense 2 for 48 h (Fig. 4), and this inhibition was positively correlated with the dose of antisense oligonucleotide. The peak value of the volume-activated current decreased from 1,431 ± 92 pA with 20 µg/ml Lipofectin alone (n = 29) to 966 ± 215 (n = 6), 740 ± 161 (n = 10; P < 0.01), 624 ± 61 (n = 13; P < 0.01), and 203 ± 81 (n = 4; P < 0.01) pA at the +80-mV step after treatments with 20 µg/ml Lipofectin and 50, 100, 200, and 400 µg/ml antisense 2, respectively. Apart from the inhibition of the peak currents, antisense 2 delayed the activation of the volume-activated Cl- currents. The latency of activation increased from 126 ± 17 s in Lipofectin alone to 163 ± 56, 212 ± 36 (P < 0.01), 243 ± 23 (P < 0.01), and 335 ± 61 s (P < 0.01) in 20 µg/ml Lipofectin plus 50, 100, 200, and 400 µg/ml antisense 2, respectively.


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Fig. 4.   Dose response of inhibition of a specific human pICln antisense oligonucleotide (antisense 2). A: whole cell currents activated by hypotonic solution. Traces represent peak currents activated by hypotonic condition and elicited by a +80-mV step in presence of Lipofectin (Lipo, 20 µg/ml) and of antisense 2 (A2) at increasing concentrations (50, 100, 200, and 400 µg/ml, each with 20 µg/ml Lipofectin). B: dose response curves for antisense 2 inhibition. Mean current elicited by a +80-mV step is plotted as a function of time before and after (solid lines) exposure and during exposure (dotted lines) to hypotonic solution beginning at arrow. Cells were incubated in 50 (black-down-triangle ), 100 (black-triangle), 200 (), and 400 (bullet ) µg/ml antisense 2 + 20 µg/ml Lipofectin or in 20 µg/ml Lipofectin alone (black-diamond ) for 48 h. Data demonstrate that latency, defined as time taken for whole cell current to be activated following exposure to hypotonic solution, increased and that peak currents decreased in a dose-dependent manner.

Antisense 1 and antisense 3, on the other hand, had no effect at any of the applied concentrations. The latency of activation and the peak currents in these groups were not significantly different from those in the Lipofectin group (Figs. 5 and 6). The latency and the peak current at the +80-mV step were 126 ± 17 s and 1,431 ± 92 pA (20 µg/ml Lipofectin; n = 29), 126 ± 42 s and 1,450 ± 189 pA (20 µg/ml Lipofectin and 200 µg/ml antisense 1; n = 5), and 140 ± 40 s and 1,421 ± 98 pA (20 µg/ml Lipofectin and 200 µg/ml antisense 3; n = 8).


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Fig. 5.   Effect of antisense 3. Recording and conditions were similar to those for Fig. 4. A: whole cell currents activated by hypotonic solution. Traces represent peak currents activated by hypotonic condition and elicited by a +80-mV step in presence of Lipofectin (20 µg/ml) and of antisense 3 (A3) at increasing concentrations (50, 100, 200, and 300 µg/ml, each with 20 µg/ml Lipofectin). B: dose response curves for antisense 3 inhibition. Mean current elicited by a +80-mV step is plotted as a function of time before and after (solid lines) exposure and during exposure (dotted lines) to hypotonic solution. Cells were incubated in 50 (black-down-triangle ), 100 (black-triangle), 200 (), and 300 (bullet ) µg/ml antisense 3 + 20 µg/ml Lipofectin or in 20 µg/ml Lipofectin alone (black-diamond ) for 48 h. Data demonstrate that antisense 3 had no effect on latency or peak currents.


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Fig. 6.   Comparison of effects of antisense 1, antisense 2, and antisense 3 (A3). A: current traces. B: time-dependent plots of mean currents. Currents were elicited by +80-mV steps. Mean current elicited by a +80-mV step is plotted as a function of time before and after (solid lines) exposure and during exposure (dotted lines) to hypotonic solution. Cells were incubated for 48 h in 20 µg/ml Lipofectin alone (black-down-triangle ), Lipofectin with 200 µg/ml antisense 1 (), Lipofectin with 200 µg/ml antisense 2 (black-triangle), and Lipofectin with 200 antisense 3 (bullet ).

Sense oligonucleotides had no effect on the volume-activated currents in four experiments.

Relationship between concentration and uptake of antisense oligonucleotides and the volume-activated Cl- current. Figure 7 shows the relationship between the antisense oligonucleotide concentration, the uptake of antisense oligonucleotides, and the volume-activated Cl- current. When the concentration of antisense 2 was increased, the uptake of antisense 2 by the cells (the fluorescence inside the cells) increased (Fig. 7A), the volume-activated Cl- current decreased (Fig. 7B), and the activation was delayed (the latency increased; Fig. 7C). There was a strong inverse correlation between antisense oligonucleotide fluorescence and mean volume-activated current (r = -0.98, P < 0.01; Fig. 7D), and there was a positive correlation between the antisense 2 fluorescence and the latency of the activation of Cl- current (r = 0.96, P < 0.01; Fig. 7E).


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Fig. 7.   Correlation between concentration of antisense oligonucleotides in culture medium, uptake of antisense oligonucleotides by NPCE cells, latency, and peak currents activated by hypotonic solution. A: uptake of antisense 2 (gray level of fluorescence) vs. concentration of antisense 2. B: mean peak currents vs. concentration of antisense 2. C: latency of activation of hypotonicity-induced Cl- current vs. concentration of antisense 2. D: mean peak current vs. uptake of antisense 2. E: latency vs. uptake of antisense 2. Dotted lines in D and E are linear regression fits to data. Latency and mean peak current were measured for cells exposed to Lipofectin (20 µg/ml) alone or to 20 µg/ml Lipofectin with 50, 100, 200, or 400 µg/ml antisense oligonucleotide for 48 h, and these values are compared with gray level of fluorescence (uptake of antisense 2) exhibited by cells exposed to same treatment regime.

pICln immunofluorescence. Figure 8 demonstrates pICln protein immunofluorescence in ciliary epithelial cells using the pICln antibody. The cells were incubated in the absence and presence of the pICln antibody in the control groups. In the absence of the antibody there was little (autofluorescence) or no fluorescence. In the presence of the antibody, pICln protein immunofluorescence was detected in the ciliary epithelial cells. The pICln protein immunofluorescence of the nonpigmented cells was stronger than that of the pigmented cells. In the immmunofluorescence experiments on antisense oligonucleotide groups, all the cells were incubated in the presence of pICln antibody. Incubation of cells in the presence of antisense 2 for 48 h caused a significant reduction of the pICln protein immunofluorescence. This reduction was evident in almost all cells examined (see Fig. 8). There was no significant effect of antisense 1 or antisense 3 on the pICln protein immunofluorescence under the same conditions.


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Fig. 8.   pICln protein immunofluorescence. Pairs (A and B, C and D, E and F, G and H) represent light and confocal fluorescence images of cells, respectively. Cells were incubated for 48 h in absence (A and B, C and D) and presence of 20 µg/ml Lipofectin with 200 µg/ml antisense 2 (E and F) or with 200 µg/ml antisense 3 (G and H) before immunofluorescence studies. For immunofluorescence experiments, cells were incubated in absence (A and B) and presence (C and D, E and F, G and H) of pICln antibody; C and D demonstrate pICln protein immunofluorescence in ciliary epithelial cells. This pICln protein immunofluorescence was knocked down by antisense 2 (E and F). Objective magnification was ×20.

Quantification of the pICln protein immunofluorescence by analysis of the confocal images of the nonpigmented cells is given in Fig. 9. The fluorescence (gray level) values are expressed in the units on an 8-bit scale in which 0 = black and 255 = white. Incubation in 200 µg/ml antisense 2 reduced the pICln protein immunofluorescence by 59%, from 34.3 ± 2.0 units in control (n = 14) to 14.0 ± 2.5 units in antisense 2 (n = 16, P < 0.01). Antisense 1 and antisense 3 had no significant effect on the pICln protein immunofluorescence. The fluorescence values in these latter two groups were 30.9 ± 4.7 (n = 13) and 36.5 ± 2.5 units (n = 17), respectively.


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Fig. 9.   Quantification of pICln protein immunofluorescence, achieved by analyzing confocal images of nonpigmented cells. Fluorescence (gray level) values are expressed in units on an 8-bit scale in which 0 = black and 255 = white. Cells were incubated in absence (CTRL0; n = 39) and presence (CTRL; n = 14) of pICln antibody and with pICln antibody and Lipofectin (20 µg/ml) together with 200 µg/ml of antisense 1 (n = 13), antisense 2 (n = 16), or antisense 3 (n = 17).

Inhibition of regulatory volume decrease by pICln antisense oligonucleotides. The volume of the cells was monitored using a light scattering technique (see Volume measurements). As cells swell, they scatter light and the reflected beam intensity decreases. In the control group (Fig. 10A; n = 4), the light intensity decreased when the cells swelled following exposure to hypotonic solution, and then intensity returned to the control level as the cells underwent regulatory volume decrease (RVD). Treating the cells with 200 µg/ml antisense 2 for 48 h (n = 4) caused a significant reduction of the RVD; the light intensity level detected did not return to control levels (Fig. 10B).


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Fig. 10.   Regulatory volume decrease (RVD). Volume changes were followed using a light reflection and light scattering technique (see METHODS). Cell swelling caused more scattering and less reflection; thus intensity (y-axis) decreased. A: as cells swelled following exposure to hypotonic solution, light intensity decreased. Light intensity returned to control levels as cells underwent RVD and returned to normal volume. Control experiments were conducted in presence of 20 µg/ml Lipofectin. Data are means of 4 experiments; printed values are means ± SE of light intensity measured at arrows in 4 experiments in each case. B: incubation of cells in 20 µg/ml Lipofectin with 200 µg/ml antisense 2 for 48 h before testing caused a reduction in ability of cell to volume regulate. This was illustrated by failure of intensity of signal to return to control levels during exposure to hypotonic shock. Data are means of 4 experiments; printed values are means ± SE of light intensity measured at arrows in 4 experiments in each case.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

For the experiments reported here, we used the antisense oligonucleotide technique to knock down the expression of intrinsic pICln in a native cell type (bovine NPCE cells) and then investigated the role of the intrinsic pICln in the activation pathway of volume-activated Cl- current and cell volume regulation. Previous studies demonstrated that antisense oligonucleotides to pICln reduces the expression of the protein ICln and suppresses cell-volume-induced activation of Cl- channels in NIH/3T3 fibroblasts (16) and that antibodies to pICln suppress swelling-induced Cl- currents in Xenopus oocytes (23). Underexpression of pICln caused a 52-fold decrease in the rate of activation of the volume-activated anion current (17).

Antisense oligonucleotide uptake by cells can be barely detected using traditional application methods (40). We used the transfection agent Lipofectin to facilitate the uptake of antisense oligonucleotides in our experiments. The data showed that the NPCE cells took up the three antisense oligonucleotides we used in a dose-dependent manner.

A widespread expression of pICln has been found in different cells and tissues by using a polyclonal antiserum raised against pICln (3). Our immunofluorescence experiments demonstrated the presence of pICln protein in the native bovine ciliary epithelial cells. These results are consistent with the observations of distribution of pICln mRNA in ciliary epithelium (39). The expression of pICln (the pICln protein fluorescence) could be specifically knocked down by a pICln mRNA antisense oligonucleotide. Of the three antisense sequences we used, only antisense 2 had any effect on the expression of pICln, the volume-activated Cl- current, and volume regulation. Both antisense 1 (30 bases; +1 to +30) and antisense 2 (15 bases; +1 to +15) are complementary to the initiation codon region of the human pICln mRNA (1) starting at the initiation bases (ATG), but the base 1 of antisense 1 mismatches the human pICln mRNA. Antisense 3 (15 bases; -3 to +12) is also complementary to the initiation region of the human pICln mRNA, but starting from the third base before the initiation bases (ATG). The bovine pICln gene has not yet been cloned, so we designed the pICln antisense oligonucleotides according to the human pICln gene sequence. The first 17 bases (+1 to +17) starting from the initiation codon, ATG, of pICln gene in human are the same as those in dog, rat, and mouse (18, 23, 32, 41), but there are some differences after that between species. The first base (-1) preceding the initiation codon (ATG) is different among human, dog, rat, and mouse. We may therefore postulate that only antisense 2 is completely complementary to the bovine pICln mRNA. This would explain why only antisense 2 was effective. The results suggest that the antisense oligonucleotide effects in our experiments are of high specificity.

After we knocked down the expression of pICln in NPCE cells by using a pICln mRNA antisense oligonucleotide, the volume-activated Cl- current decreased, its activation was delayed, and the extent of RVD following cell swelling was diminished. The data demonstrated that the intrinsic pICln plays an important role in the activation pathway of volume-activated Cl- current and cell volume regulation. In our experiments, treatment with antisense 2 prolonged the activation time (latency) of the Cl- current. The increase in latency of activation was not due to different rates of swelling; the time constants for swelling were 15.3 and 14.6 s for control and antisense oligonucleotide-treated cells, respectively. This suggests that pICln protein may function as a Cl- channel regulator. However, we cannot exclude the possibility that the pICln protein functions additionally as a channel, because the value of the current also decreased. Several commentators recently published their belief that pICln is not the swelling-activated Cl- channel (5, 34) and may not necessarily be directly involved with either the swelling-activated Cl- current or volume homeostasis (34). This view is hard to reconcile with the data presented in this paper. There are significant concerns raised by the cytosolic localization of pICln, as discussed in the introduction, and the precise role of pICln awaits further elucidation.

Besides pICln, previous work in our laboratory has demonstrated that P-glycoprotein is involved in the activation of volume-activated Cl- current in the bovine NPCE cell (42). It was also reported that ClC-3 was associated with a volume-activated Cl- current, and a scheme was presented in which pICln, shown to be expressed in cloned ciliary epithelial cells (1), linked actin to the opening of ClC-3 Cl- channels (7). What then is the relationship between these three proteins? Are they different channels or do they work cooperatively as a single system associated with the volume-activated Cl- current? More work must be done to answer these questions.

It has been suggested that the same mechanisms that are responsible for cell volume regulation are recruited for the secretion of aqueous humor (12, 43), and Cl- channels have been hypothesized to be the rate-limiting factor in the formation of the aqueous humor (6). Our experiments demonstrate that pICln is present in the native ciliary epithelial cells and that pICln plays an important role in the activation pathway of volume-activated Cl- current and cell volume regulation. These findings suggest that the pICln protein may be involved in the secretion of aqueous humor.

    ACKNOWLEDGEMENTS

We are indebted to Prof. Marcus Paulmichl for the gift of pICln antibody.

    FOOTNOTES

The Royal National Institute for the Blind and The Medical Research Council supported this work.

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. §1734 solely to indicate this fact.

Address for reprint requests: T. J. C. Jacob, PO Box 911, School of Biosciences, Cardiff University, Cardiff CF1 3US, United Kingdom.

Received 1 July 1998; accepted in final form 2 October 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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