Association of intrinsic pICln with volume-activated Cl2
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
antisense oligonucleotides; ciliary epithelium; secretion; fluid
transport; ion channels; nonpigmented ciliary epithelial cells
Cl2 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 Cl2 channels are considered
to be involved in the formation of aqueous humor (20).
During secretion, the main anion secreted is Cl2 (4, 24),
and Cl2 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 Cl2 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 Cl2 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
A VOLUME-ACTIVATED
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C182
the activation of volume-activated Cl2 current. CocaPrados and colleagues (7) showed that the NPCE cells
express transcripts for ClC-3 and suggested that ClC-3
was the volume-activated Cl2 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 Cl2 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 volumeactivated Cl2 current? In this study, we demonstrate
the presence of pICln in a native cell, its association with
the volume-activated Cl2 current, and its involvement
with cell volume regulation.
METHODS
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 Ca21- and Mg21-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.
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
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Chen, Lixin, Liwei Wang, and Tim J. C. Jacob. Association of intrinsic pICln with volume-activated Cl2 current and
volume regulation in a native epithelial cell. Am. J. Physiol.
276 (Cell Physiol. 45): C182–C192, 1999.—We investigated
the relationship between pICln, the volume-activated Cl2
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 Cl2
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 Cl2 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 Cl2 current and
RVD following hypotonic swelling.
PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
Kent, UK) on a two-stage vertical puller (PB-7, Narishige,
Tokyo, Japan) and gave a resistance of 5–10 MV 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 Cl2 equilibrium potential (0 mV) and then polarized to 240, 140, 0, 280, and 180
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 Cl2 currents. The pipette solution contained (in mM) 105 N-methylD-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 Lipo-
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,
320.
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Oligonucleotides. The four HPLC-purified oligonucleotides
were synthesized by Severn Biotech (Milton Keynes, UK).
The sense sequence, from base 23 to base 112, was 58-GCT
ATG AGC TTC CTC-38. The sequences of antisense 1, 58-CGG
CGG CGG GAA ACT TTT GAG GAA GCT CAT-38, and
antisense 2, 58-TTT GAG GAA GCT CAT-38, are complementary to the initiation codon region of the human pICln mRNA
(1) starting at the initiation codon. The sequence of antisense
3, 58-GAG GAA GCT CAT AGC-38, 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 serumfree 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,
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PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
fectin, 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 5 black and 255 5
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
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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 5 black, 255 5 white) measured by confocal microscopy.
There was no significant difference in fluorescence between control
(CTRL; no additives; n 5 18) and Lipofectin alone (20 µg/ml; n 5 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.
PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
RESULTS
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 6 7.7 units (24 h; n 5 17) and 61.9 6
5.5 units (48 h; n 5 15) in antisense 1 (200 µg/ml; Fig.
2A), 54.9 6 4.6 units (24 h; n 5 20) and 50.4 6 4.6 units
(48 h; n 5 32) in antisense 2 (200 µg/ml; Fig. 2B), and
78.2 6 7.6 units (24 h; n 5 22) and 77.3 6 7.7 units
(48 h; n 5 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 receptorlike recognition, and it may depend on such factors as
oligonucleotide chain length and class (38).
Volume-activated Cl2 current. The whole cell currents in single NPCE cells were recorded using patchclamp technique 48 h after treatment with oligonucleotide and/or Lipofectin. Figure 3 illustrates the whole
cell current in response to voltage steps of 240, 140, 0,
280, and 180 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 240, 140, 0, 280, and 180
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
Fig. 3. Whole cell voltage-clamp recordings from single
NPCE cells. Patch pipette contained a buffer designed
for examination of Cl2 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 (240, 140, 0, 280, and 180
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.
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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 6 SE (n is the
number of observations) and where appropriate were analyzed using Student’s t-test.
C185
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PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
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 180-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 180-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 (m), 100 (l), 200 (j), and 400 (r) µg/ml antisense 2 1 20 µg/ml
Lipofectin or in 20 µg/ml Lipofectin alone (p) 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.
of antisense oligonucleotides, and the volume-activated
Cl2 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 Cl2 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 5 20.98, P , 0.01; Fig. 7D),
and there was a positive correlation between the antisense 2 fluorescence and the latency of the activation of
Cl2 current (r 5 0.96, P , 0.01; Fig. 7E).
pICln immunofluorescence. Figure 8 demonstrates pICln
protein immunofluorescence in ciliary epithelial cells
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a voltage that was close to the equilibrium potential for
Cl2 (Fig. 3C). The bathing solution was then changed
back to isotonic solution 7 min after the activation of
the Cl2 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 Cl2 (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.
In the control group (no additives), the latency of the
activation of the Cl2 currents was 124 6 29 s (n 5 11),
after which the currents increased. The peak current
elicited by a step of 180 mV (taken at 7 min after
latency) was 1,599 6 116 pA (n 5 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 6
17 s and 1,431 6 92 pA (n 5 29), respectively, at the
180-mV step.
pICln antisense oligonucleotides inhibited the activation of volume-activated current. The volume-activated
Cl2 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 6 92 pA with 20 µg/ml
Lipofectin alone (n 5 29) to 966 6 215 (n 5 6), 740 6
161 (n 5 10; P , 0.01), 624 6 61 (n 5 13; P , 0.01), and
203 6 81 (n 5 4; P , 0.01) pA at the 180-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 Cl2 currents. The
latency of activation increased from 126 6 17 s in
Lipofectin alone to 163 6 56, 212 6 36 (P , 0.01), 243 6
23 (P , 0.01), and 335 6 61 s (P , 0.01) in 20 µg/ml
Lipofectin plus 50, 100, 200, and 400 µg/ml antisense 2,
respectively.
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 180-mV step were 126 6 17 s and
1,431 6 92 pA (20 µg/ml Lipofectin; n 5 29), 126 6 42 s
and 1,450 6 189 pA (20 µg/ml Lipofectin and 200 µg/ml
antisense 1; n 5 5), and 140 6 40 s and 1,421 6 98 pA
(20 µg/ml Lipofectin and 200 µg/ml antisense 3; n 5 8).
Sense oligonucleotides had no effect on the volumeactivated currents in four experiments.
Relationship between concentration and uptake of
antisense oligonucleotides and the volume-activated
Cl2 current. Figure 7 shows the relationship between
the antisense oligonucleotide concentration, the uptake
PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
C187
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 180-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 180-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 (m), 100 (l), 200 (j), and 300 (r) µg/ml
antisense 3 1 20 µg/ml Lipofectin or in 20 µg/ml Lipofectin alone (p)
for 48 h. Data demonstrate that antisense 3 had no effect on latency or
peak currents.
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.
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 180-mV steps. Mean current
elicited by a 180-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 (m), Lipofectin with 200 µg/ml antisense 1 (j),
Lipofectin with 200 µg/ml antisense 2 (l), and Lipofectin with 200
antisense 3 (r).
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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 5 black and 255 5 white. Incubation in 200
µg/ml antisense 2 reduced the pICln protein immunofluorescence by 59%, from 34.3 6 2.0 units in control (n 5
14) to 14.0 6 2.5 units in antisense 2 (n 5 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 6 4.7 (n 5
13) and 36.5 6 2.5 units (n 5 17), respectively.
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
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PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
and the reflected beam intensity decreases. In the
control group (Fig. 10A; n 5 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 5 4) caused a significant
reduction of the RVD; the light intensity level detected
did not return to control levels (Fig. 10B).
DISCUSSION
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 volumeactivated Cl2 current and cell volume regulation. Previ-
ous studies demonstrated that antisense oligonucleotides to pICln reduces the expression of the protein ICln
and suppresses cell-volume-induced activation of Cl2
channels in NIH/3T3 fibroblasts (16) and that antibodies to pICln suppress swelling-induced Cl2 currents in
Xenopus oocytes (23). Underexpression of pICln caused a
52-fold decrease in the rate of activation of the volumeactivated 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
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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 Cl2 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
AND VOLUME-ACTIVATED CL2 CURRENTS
C189
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 5 black and 255 5 white. Cells were incubated in absence
(CTRL0; n 5 39) and presence (CTRL; n 5 14) of pICln antibody and
with pICln antibody and Lipofectin (20 µg/ml) together with 200
µg/ml of antisense 1 (n 5 13), antisense 2 (n 5 16), or antisense 3 (n 5
17).
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 Cl2 current, and
volume regulation. Both antisense 1 (30 bases; 11 to
130) and antisense 2 (15 bases; 11 to 115) 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; 23 to 112) 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 (11 to 117) 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 (21) 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 oligo-
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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 320.
C190
PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
We are indebted to Prof. Marcus Paulmichl for the gift of pICln
antibody.
The Royal National Institute for the Blind and The Medical
Research Council supported this work.
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.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017
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 6 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 6 SE of
light intensity measured at arrows in 4 experiments in each case.
nucleotide 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 2 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
Cl2 current and cell volume regulation. In our experiments, treatment with antisense 2 prolonged the activation time (latency) of the Cl2 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 Cl2 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 Cl2 channel (5, 34) and may not
necessarily be directly involved with either the swellingactivated Cl2 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 Cl2 current in the bovine NPCE cell (42). It was also reported that ClC-3
was associated with a volume-activated Cl2 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 Cl2 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 volumeactivated Cl2 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 Cl2 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 Cl2 current and cell volume regulation. These findings suggest that the pICln protein may
be involved in the secretion of aqueous humor.
PICLN
AND VOLUME-ACTIVATED CL2 CURRENTS
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