School of Biosciences, Cardiff University, Cardiff CF1 3US, United Kingdom
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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 M 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).
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.
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RESULTS |
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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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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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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We are indebted to Prof. Marcus Paulmichl for the gift of pICln antibody.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anguita, J.,
M. L. Chalfant,
M. M. Civan,
and
M. Coca-Prados.
Molecular cloning of the human volume-sensitive chloride conductance regulatory protein, from ocular ciliary epithelium.
Biochem. Biophys. Res. Commun.
208:
89-95,
1995[Medline].
2.
Arreola, J.,
J. E. Melvin,
and
T. Begenisch.
Volume-activated chloride channels in rat parotid acinar cells.
J. Physiol. (Lond.)
484:
677-687,
1995[Abstract].
3.
Buyes, G.,
C. D. Greef,
L. Raeymaekers,
G. Droogmans,
B. Nilius,
and
J. Eggermont.
The ubiquitously expressed pICln protein forms homomeric complexes in vitro.
Biochem. Biophys. Res. Commun.
218:
822-827,
1996[Medline].
4.
Caprioli, J.
The ciliary epithelia and aqueous humor.
In: Alder's Physiology of Eye: Clinical Application, edited by R. A. Moses,
and W. M. Hart, Jr.. St. Louis, MO: Mosby, 1987, p. 204-222.
5.
Clapham, D. E.
The list of potential volume-sensitive chloride currents continues to swell (and shrink).
J. Gen. Physiol.
111:
623-624,
1998
6.
Coca-Prados, M.,
J. Anguita,
M. L. Chalfant,
and
M. M. Civan.
PKC-sensitive Cl channels associated with ciliary epithelial homologue of pICln.
Am. J. Physiol.
268 (Cell Physiol. 37):
C572-C579,
1995
7.
Coca-Prados, M.,
J. Sánchez-Torres,
K. Peterson-Yantorno,
and
M. M. Civan.
Association of ClC-3 channel with Cl transport by human nonpigmented ciliary epithelial cells.
J. Membr. Biol.
150:
197-208,
1996[Medline].
8.
Diaz, M.,
M. A. Valverde,
C. F. Higgins,
C. Rucareanu,
and
F. V. Sepulveda.
Volume-activated chloride channels in HeLa cells are blocked by verapmil and dideoxyforskolin.
Pflügers Arch.
422:
347-353,
1993[Medline].
9.
Doroshenko, P.,
and
E. Neher.
Volume-sensitive conductance in bovine chromaffin cell membrane.
J. Physiol. (Lond.)
449:
197-218,
1992[Abstract].
10.
Duan, D.,
C. Winter,
S. Cowley,
J. R. Hume,
and
B. Horowitz.
Molecular identification of a volume-regulated chloride channel.
Nature
390:
417-421,
1997[Medline].
11.
Emma, F.,
S. Breton,
R. Morrison,
S. Wright,
and
K. Strange.
Effect of cell swelling on membrane and cytoplasmic distribution of pICln.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1545-C1551,
1998
12.
Farahbakhsh, N. A.,
and
G. L. Fain.
Volume regulation of non-pigmented cells from ciliary epithelium.
Invest. Ophthalmol. Vis. Sci.
28:
934-944,
1987[Abstract].
13.
Gill, D. R.,
S. C. Hyde,
C. F. Higgins,
M. A. Valverde,
G. M. Mintenig,
and
F. V. Sepulveda.
Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein.
Cell
71:
23-32,
1992[Medline].
14.
Goldstein, L.,
E. M. Davis-Amaral,
H. H. Vandeburgh,
and
M. W. Musch.
Hypotonic exposure stimulates translocation of the swelling-activated channel protein pICln in neonatal rat myocytes (Abstract).
J. Gen. Physiol.
111:
36a,
1997.
15.
Grunder, S.,
A. Thiemann,
M. Pusch,
and
T. J. Jentsch.
Regions involved in the opening of ClC-2 chloride channel by voltage and cell volume.
Nature
360:
759-762,
1992[Medline].
16.
Gschwentner, M.,
U. O. Nagl,
E. Woll,
A. Schmarda,
M. Ritter,
and
M. Paulmichl.
Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.
Pflügers Arch.
430:
464-470,
1995[Medline].
17.
Hubert, M. D.,
I. Levitan,
and
S. S. Garber.
Activation of volume-regulated anion current (VRAC) is dependent on ICln expression (Abstract).
Biophys. J.
74:
A221,
1998.
18.
Ishibashi, K.,
S. Sasaki,
S. Uchida,
T. Imai,
and
F. Marumo.
Tissue expression of mRNA of chloride channel from MDCK cells and its regulation by protein kinases.
Biochem. Biophys. Res. Commun.
192:
561-567,
1993[Medline].
19.
Jacob, T. J. C.
The identification of a low-threshold, T-type calcium channel in bovine ciliary epithelial cells.
Am. J. Physiol.
261 (Cell Physiol. 30):
C808-C813,
1991
20.
Jacob, T. J. C.,
and
M. M. Civan.
Role of ion channels in aqueous humor formation.
Am. J. Physiol.
271 (Cell Physiol. 40):
C703-C720,
1996
21.
Kelly, M. E. M.,
S. J. Dixon,
and
S. M. Sims.
Outwardly rectifying chloride current in rabbit osteoclasts is activated by hyposmotic stimulation.
J. Physiol. (Lond.)
475:
377-389,
1994[Abstract].
22.
Kotera, T.,
and
P. D. Brown.
Calcium-dependent chloride current activated by hyposmotic stress in rat lacrimal acinar cells.
J. Membr. Biol.
134:
67-74,
1993[Medline].
23.
Krapivinsky, G. B.,
M. J. Ackerman,
E. A. Gordon,
L. D. Krapivinsky,
and
D. E. Clapham.
Molecular characterization of a swelling-induced chloride conductance regulatory protein, pICln.
Cell
76:
439-448,
1994[Medline].
24.
Krupin, T.,
and
M. M. Civan.
The physiologic basis of aqueous humor formation.
In: The Glaucomas (2nd ed.), edited by R. Ritch,
M. B. Shields,
and T. Krupin. St. Louis, MO: Mosby, 1995, p. 251-280.
25.
Kubo, M.,
and
Y. Okada.
Volume-regulatory Cl channel currents in cultured human epithelial cells.
J. Physiol. (Lond.)
456:
351-371,
1992[Abstract].
26.
Kunzelmann, K.,
R. Kubitz,
M. Glolik,
R. Wart,
and
R. Greger.
Small-conductance Cl channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP.
Pflügers Arch.
421:
238-246,
1992[Medline].
27.
Laich, A.,
U. O. Furst,
M. Nagl,
M. Gschwentner,
and
M. Paulmichl.
The transposition of the swelling-dependent chloride channel pICln from the cytosol to the membrane is regulated by cell volume (Abstract).
J. Gen. Physiol.
108:
24a,
1996.
28.
Lewis, R. S.,
P. E. Ross,
and
M. D. Cahalan.
Chloride channels activated by osmotic stress in T lymphocytes.
J. Gen. Physiol.
101:
801-826,
1993[Abstract].
29.
Musch, M. W.,
C. A. Luer,
E. M. Davis-Amaral,
and
L. Goldstein.
Hypotonic stress induces translocation of the osmolyte channel protein pICln in embryonic skate.
J. Exp. Zool.
277:
460-463,
1997[Medline].
30.
Nilius, B.,
M. Oike,
I. Zahradnik,
and
G. Droogmans.
Activation of a Cl current by hypotonic volume increase in human endothelial cells.
J. Gen. Physiol.
103:
787-805,
1994[Abstract].
31.
Nilius, B.,
J. Sehrer,
F. Viana,
C. D. Greef,
L. Raeymaekers,
J. Eggermont,
and
G. Droogmans.
Volume-activated Cl currents in different mammalian non-excitable cell types.
Pflügers Arch.
428:
364-371,
1994[Medline].
32.
Paulmichl, M.,
Y. Li,
K. Wickman,
M. Ackerman,
E. Peralta,
and
D. Clapham.
New mammalian chloride channel identified by expression cloning.
Nature
356:
238-241,
1992[Medline].
32a.
Schwartz, R. S.,
A. C. Rybicki,
and
R. L. Nagel.
Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.
Biochem. J.
327:
609-616,
1997[Medline].
33.
Solc, C. K.,
and
J. J. Wine.
Swelling-induced and depolarization-induced Cl channels in normal and cystic fibrosis epithelial cells.
Am. J. Physiol.
261 (Cell Physiol. 30):
C658-C674,
1991
34.
Strange, K.
Molecular identity of the outwardly rectifying, swelling-activated anion channel: time to reevaluate pICln.
J. Gen. Physiol.
111:
617-622,
1998
35.
Strange, K.,
F. Emma,
and
P. S. Jackson.
Cellular and molecular physiology of volume-sensitive anion channels.
Am. J. Physiol.
270 (Cell Physiol. 39):
C711-C730,
1996
37.
Valverde, M. A.,
M. Diaz,
M. Sepulveda,
D. G. Gill,
S. C. Hyde,
and
C. F. Higgins.
Volume regulated chloride channels are associated with the human multidrug-resistance P-glycoprotein.
Nature
355:
830-833,
1992[Medline].
38.
Wahlestedt, C.
Antisense oligonucleotide strategies in neuropharmacology.
Trends Pharmacol. Sci.
15:
42-46,
1994[Medline].
39.
Wan, X. L.,
S. Chen,
and
M. Sears.
Cloning and functional expression of a swelling-induced chloride conductance regulatory protein, pICln, from rabbit ocular ciliary epithelium.
Biochem. Biophys. Res. Commun.
239:
692-696,
1997[Medline].
40.
Wang, L.,
L. Chen,
V. E. Walker,
and
T. J. C. Jacob.
Antisense to MDR1 mRNA reduces P-glycoprotein expression, swelling-activated Cl current and volume regulation in bovine ciliary epithelial cells.
J. Physiol. (Lond.)
511:
33-44,
1998
41.
Wickman, K.,
M. F. Seldin,
M. R. James,
S. J. Gendler,
and
D. E. Clapham.
Partial structure, chromosome localization, and expression of the mouse ICln gene.
Genomics
40:
402-408,
1997[Medline].
42.
Wu, J.,
J. J. Zhang,
H. Koppel,
and
T. J. C. Jacob.
P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells.
J. Physiol. (Lond.)
491:
743-755,
1996[Abstract].
43.
Yantorno, R. E.,
D. A. Carré,
M. Coca-Prados,
T. Krupin,
and
M. M. Civan.
Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling.
Am. J. Physiol.
262 (Cell Physiol. 31):
C501-C509,
1992
44.
Zhang, J. J.,
and
T. J. C. Jacob.
Three different Cl channels in the bovine ciliary epithelium activated by hypotonic stress.
J. Physiol. (Lond.)
499:
379-389,
1997[Abstract].