1 Center of Biomedical Research Excellence, Department of Pharmacology, University of Nevada, Reno, Nevada 89557-0046; and 2 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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We tested the possible
role of endogenous protein kinase C (PKC) in the regulation of native
volume-sensitive organic osmolyte and anion channels (VSOACs) in
acutely dispersed canine pulmonary artery smooth muscle cells (PASMC).
Hypotonic cell swelling activated native volume-regulated
Cl currents (ICl.vol) which could
be reversed by exposure to phorbol 12,13-dibutyrate (0.1 µM) or by
hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 µM) or Ro-31-8425 (0.1 µM), inhibitors of both conventional
and novel PKC isozymes, significantly activated
ICl.vol and prevented further modulation by
subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 µM), a
selective conventional PKC inhibitor, was without effect. Dialyzing
acutely dispersed and cultured PASMC with
V1-2 (10 µM), a
translocation inhibitory peptide derived from the V1 region of
PKC,
activated ICl.vol under isotonic conditions and
prevented further modulation by cell volume changes. Dialyzing PASMC
with
C2-2 (10 µM), a translocation inhibitory peptide derived
from the C2 region of
PKC, had no detectable effect.
Immunohistochemistry in cultured canine PASMC verified that hypotonic
cell swelling is accompanied by translocation of
PKC from the
vicinity of the membrane to cytoplasmic and perinuclear locations.
These data suggest that membrane-bound
PKC controls the activation
state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.
chloride channels; cell volume; protein kinase C
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INTRODUCTION |
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VOLUME-SENSITIVE ORGANIC
OSMOLYTE and anion channels (VSOACs) are ubiquitously expressed
in mammalian cells and play an important physiological role in a
variety of diverse cellular functions, including the regulation of
electrical activity, cell volume homeostasis, intracellular pH,
immunological responses, cell proliferation, and differentiation
(24, 27, 34). The cellular and molecular mechanisms that
link changes in cell volume to the activation of volume-sensitive
Cl currents (ICl.vol) are not well
understood. Although changes in intracellular ionic strength have been
proposed as the initial trigger for activation of
ICl.vol in some cell types (11,
37), reorganization of the F-actin cytoskeleton during cell
volume changes may result in changes in membrane physical properties or
alterations of the properties or distribution of many proteins normally
associated with the cell membrane. It has been suggested that membrane
unfolding may be the stimulus for activation of ICl.vol and that the F-actin cytoskeleton may
modulate volume sensitivity of the channel by conferring resistance to
this process (27). It has recently been shown that
caveolins, the principal protein of membrane caveolae, modulate the
properties of ICl.vol (36). A
number of protein kinases, which have been shown to play a role in cell
volume regulation, including tyrosine kinase (35, 38),
Rho-associated kinase (25), phosphatidylinositol (PI)
3-kinase (12), and protein kinase C (PKC) (2,
6), have all been shown to directly modulate the activity of
ICl.vol in a variety of different native cell
types. However, a clear picture of the actual cascade of intracellular
events linking cell volume changes to the regulation of
ICl.vol has yet to emerge. It is not even clear
whether or not a single unifying mechanism for activation of
ICl.vol necessarily applies to all cells studied or whether different protein entities may be responsible for
ICl.vol in different cell types (7,
34).
We previously tested the hypothesis that PKC-catalysed phosphorylation and dephosphorylation by protein phosphatases may represent an important molecular link between changes in cell volume and ICl.vol regulation in native cardiac myocytes, Xenopus oocytes, and the recent molecular candidate for VSOAC, guinea pig ClC-3 (gpClC-3) expressed in NIH/3T3 cells (5, 8, 32). Hypotonic cell swelling was shown to activate, whereas hypertonic cell shrinkage was shown to deactivate, gpClC-3 currents (IgpClC-3) expressed in NIH/3T3 cells and ICl.vol in native cardiac myocytes and Xenopus oocytes, effects that could be mimicked under isotonic conditions by inhibition (bisindolylmaleimide; BIM) and stimulation (phorbol esters), respectively, of endogenous PKC. Moreover, phosphatase inhibitors such as okadaic acid and calyculin A also inhibited Igp-ClC-3, and mutation of an amino terminal PKC phosphorylation site (S51A) completely eliminated the response of expressed IgpClC-3 to cell swelling, resulting in a constitutively active channel under isotonic conditions which was insensitive to PKC activation and phosphatase inhibition. These results indicated that an important mechanism linking cell swelling to activation of ICl.vol in native cardiac myocytes, Xenopus oocytes, and recombinant IgpClC-3 involves dephosphorylation of PKC-phosphorylated sites.
The properties of ICl.vol have recently been studied in vascular smooth muscle cells of canine pulmonary artery, a cell type known to express endogenous ClC-3 (39), as well as rabbit portal vein (15). The purpose of the present study was to 1) determine whether or not native ICl.vol in pulmonary artery smooth muscle cells (PASMC) is regulated by PKC in a manner consistent with regulation of recombinant IgpClC-3, 2) determine which PKC isozymes are involved in the regulation of native ICl.vol by changes in cell volume in PASMC, and 3) determine whether or not the cellular distribution of PKC isozymes in PASMC is altered by changes in cell volume. A preliminary report describing these results has been published (41).
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METHODS |
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Cell preparation.
Mongrel dogs were anesthetized with pentobarbital sodium (45-50 mg
kg1, iv). Segments of pulmonary artery were removed, and
vascular smooth muscle cells were enzymatically dispersed as previously described (9, 39). Freshly dispersed cells were kept in
the refrigerator and used within 10 h. The animal use protocol was reviewed and approved by the Animal Care and Use Committee of the
University of Nevada.
Electrophysiological recordings.
Membrane currents were measured from canine PASMC at room temperature
(22-24°C) by the tight-seal, whole cell, voltage-clamp technique
(16). Patch pipettes were made from borosilicate glass capillaries and had a tip resistance of 2-5 M. Ag-AgCl wires were immersed in the bath and pipette solutions and connected to a
patch-clamp amplifier (Axopatch-200A, Axon Instruments, Foster City,
CA). A 3 M KCl-agar salt bridge between the bath and Ag-AgCl reference
electrode was used to minimize changes in liquid junctional potential.
To obtain Cl
current-voltage relations, whole cell
currents were recorded during voltage pulses (150 ms) applied from the
holding potential (
10 mV). The time courses of changes in membrane
currents were monitored by the application of repetitive voltage-clamp
steps to ±80 mV applied every 30 s. Membrane currents were
filtered at a frequency of 1 kHz and digitized online at 5 kHz using a Pentium III processor and pCLAMP 8 software (Axon Instruments).
Solutions and reagents.
All bath and pipette solutions were chosen to facilitate
Cl current recording. The standard isotonic bath solution
contained (in mM) 107 N-methyl-D-glucamine, 107 HCl, 1.5 MgCl2, 2.5 MnCl2, 10 glucose, 70 D-mannitol, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.4, 300 mosmol/kgH2O).
D-mannitol was removed from this solution to make the
standard hypotonic solution (230 mosmol/kgH2O), and 140 mM
D-mannitol was included to make the standard hypertonic
solution (370 mosmol/kgH2O). GdCl3 (0.05 mM) was routinely included in all bath solutions to prevent possible contamination of membrane currents by activation of nonselective cation
channels. The pipette solution contained (in mM) 95 CsCl, 20 tetraethylammonium chloride, 5 ATP-Mg, 5 ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid
(EGTA), 80 D-mannitol, and 5 HEPES (pH 7.2, 300 mosmol/kgH2O).
Immunohistochemistry.
PASMC were dispersed and cultured as previously described
(40). Special culture dishes were incorporated to aid with
imaging. Holes were cut (23 mm) in the base of 35-mm culture dishes,
and 25-mm glass coverslips were then bonded to the base of the dish. Cultured PASMC (48 h) were then exposed to isotonic (300 mosmol/kgH2O) and hypotonic (230 mosmol/kgH2O)
solutions for 5, 10, and 20 min. After treatment, cells were fixed with
4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) for 10 min at 4°C. After fixation, cells were washed in PBS (4 × 30 min), and nonspecific antibody binding was reduced by blocking with
10% bovine serum albumin in PBS containing 0.03% Triton X-100 for
1 h and then incubated with a polyclonal anti-PKC antibody
raised in rabbit (Upstate Biotechnology, Lake Placid, NY) at a dilution
of 1:200 for 24 h at 4°C. Cells were then washed with PBS
(2 × 10 min) and incubated in Alexa 488 (green fluorescence)
conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes)
at 5 µg/ml. Secondary incubations were performed for 1 h at room
temperature. After secondary incubation, cells were washed in PBS
(3 × 15 min). For negative control cells, the primary antibody
was omitted and substituted with PBS. Cells were examined using a Nikon
Eclipse TE 300 inverted fluorescent microscope with excitation
wavelength appropriate for Alexa 488 (488 nm). Digital micrographs were
acquired using a Spot RT digital camera, and final images were prepared using Adobe Photoshop software.
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RESULTS |
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Effect of PDBu on native ICl.vol in canine PASMC.
ICl.vol in canine PASMC are outwardly rectifying
membrane currents, with an anion permeability sequence of
SCN > I
> Br
> Cl
> aspartate
, and are inhibited
by DIDS, extracellular ATP, and the anti-estrogen compound tamoxifen
(39). Membrane currents are typically very small in
isotonic solutions (300 mosmol/kgH2O) but increase after cell swelling induced by exposure to hypotonic solutions (230 mosmol/kgH2O, Fig.
1A). Cell swelling-induced
increases in ICl.vol were completely reversed by
perfusion of cells with a hypertonic (370 mosmol/kgH2O)
solution. Figure 1B illustrates that, in a group of cells,
current densities measured at ±80 mV were more than doubled after
hypotonic challenge and returned to the basal level in response to
exposure to hypertonic bath solutions. In another group of cells, when
the activation of ICl.vol in hypotonic solutions
reached a steady state, 100 nM PDBu was applied to the hypotonic bath
solution. As shown in Fig. 1, C and D, activation of PKC by PDBu effectively reversed the hypotonic cell swelling-induced current increase. The currents after PDBu application under hypotonic conditions were no longer different from the values under isotonic conditions, although hypotonic cell swelling alone significantly increased the currents. These data suggest that, similar to native ICl.vol in cardiac myocytes, Xenopus
oocytes, and expressed ClC-3 channels in NIH 3T3 cells (5,
32), native ICl.vol in PASMC is strongly
inhibited by endogenous PKC activation.
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Effects of PKC inhibitors on native ICl.vol in canine
PASMC.
We next tested whether inhibition of endogenous PKC would cause
activation of ICl.vol in PASMC under isotonic
conditions, as previously demonstrated for native
ICl.vol in cardiac myocytes, Xenopus
oocytes, and expressed ClC-3 channels in NIH 3T3 cells (5,
32). Figure 2 shows the effect of
the PKC inhibitor, BIM at 100 nM, on ICl.vol.
BIM at this concentration is a more potent inhibitor of conventional
(c)PKCs than novel (n)PKCs (20). Unlike native
ICl.vol in cardiac myocytes and expressed ClC-3 channels in NIH 3T3 cells (5), exposure to 100 nM BIM
failed to activate ICl.vol under isotonic
conditions in PASMC. Current amplitudes after BIM exposure were not
significantly different from the values without BIM (Fig.
2B). ICl.vol in cells treated with
BIM were similar to those in control cells, being significantly increased under hypotonic conditions and reversed by exposure to
hypertonic solutions. On the other hand, exposure of PASMC to another
PKC inhibitor, calphostin C, which at 100 nM is reported to be an
equally potent inhibitor of cPKCs and nPKCs (14),
increased ICl.vol under isotonic conditions
(Fig. 3). Mean current densities in a
group of cells was more than doubled by addition of calphostin C to the
isotonic bath solution, whereas the current-voltage relationship of the
calphostin C-induced current was unchanged by switching to hypotonic
solutions (Fig. 3B). Superfusion of cells with hypotonic solutions after treatment with calphostin C failed to further increase
the current densities. Similarly, another equally potent inhibitor of
cPKCs and nPKCs, Ro-31 8425 (14), also increased ICl.vol densities under isotonic conditions and
prevented further activation of the channels by subsequent hypotonic
cell swelling (Fig. 4).
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Effects of isozyme-specific PKC translocation inhibitory peptides
on native ICl.vol in canine PASMC.
To further test the hypothesis that nPKCs are responsible for the cell
volume sensitivity of native ICl.vol in PASMC,
we examined the effects of different isozyme-specific PKC translocation
inhibitory peptides on these channels. V1-2 is a short peptide
derived from the V1 region of
PKC, which inhibits translocation of
PKC (19, 21). Figure 5
shows the effect of
V1-2 on native
ICl.vol in PASMC. Cells were dialyzed with
V1-2 (10 µM) under isotonic conditions and subsequently
exposed to hypotonic and then hypertonic solutions. Dialyzing the cells
with
V1-2 significantly increased
ICl.vol under isotonic conditions and abolished
further regulation by either hypotonic cell swelling or hypertonic cell
shrinkage (Fig. 5, A and B). The mean current
densities in cells dialyzed with
V1-2 were increased 239 ± 16% at +80 mV and 100 ± 8% at
80 mV under isotonic
conditions, and the current densities measured at 10 min of
intracellular dialysis in isotonic solutions were not significantly
different from the values after superfusion with hypotonic or
hypertonic solution. On the other hand, in cells dialyzed with the
scrambled
V1-2 peptide (10 µM), current amplitudes were
unchanged under isotonic conditions. In addition, cells dialyzed with
scrambled
V1-2 peptide responded in the usual manner to hypotonic and hypertonic solutions (Fig. 5, C and
D).
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Alterations in cellular distribution of PKC in response to cell
swelling.
Although several different PKC isoforms have been shown to
translocate in response to agonist stimulation in canine PASMC (4), the effects of changes in cell volume on PKC isoform
cellular distribution have not been previously tested. We performed
immunohistochemistry on cultured canine PASMC to examine
PKC-like
immunoreactivity in cells exposed to isotonic extracellular solutions
and after hypotonic cell swelling. Before performing these experiments, we first confirmed that dialyzing cultured PASMC with the
V1-2 peptide produced similar effects on ICl.vol as
previously observed in acutely dispersed PASMC (Fig.
7). Figure
8A illustrates the typical
pattern of immunoreactivity of a canine PASMC under isotonic conditions.
PKC-like immunoreactivity showed a diffuse, uniform cytosolic and membrane disposition. This is illustrated in Fig. 8B in the plot of pixel intensity over the region of
interest illustrated (rectangular box) in Fig. 8A. In
contrast, there appeared to be a consistent decrease in near-membrane
immunoreactivity (horizontal dashed lines) and a marked increase in
cytoplasmic and perinuclear
PKC-like immunoreactivity in cells
exposed to hypotonic solutions for 20 min (Fig. 8, C and
D). These differences were quantitated by measuring mean
pixel intensity in areas juxtaposed near the sarcolemmal membrane or
near the nuclear membrane and compared in cells exposed to either
isotonic or hyotonic bath solutions (Fig. 8D).
PKC-like
immunoreactivity in regions near the sarcolemmal membrane were
significantly lower in cells exposed to hypotonic solutions compared
with similar regions in cells exposed to isotonic solutions. There also
was a significant increase in
PKC-like immunoreactivity in
perinuclear regions in cells exposed to hypotonic solutions compared
with similar regions in cells exposed to isotonic solutions. These data
suggest that hypotonic cell swelling is associated with a translocation
of
PKC from the vicinity of the sarcolemmal membrane to the
cytoplasm and perinuclear regions.
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DISCUSSION |
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For ICl.vol in native cardiac myocytes,
Xenopus oocytes, and recombinant ClC-3 Cl
channels expressed in NIH 3T3 cells, channels can be activated under
isotonic conditions by exposure of cells to 100 nM BIM, a highly
selective inhibitor of cPKCs (20), whereas stimulation of
endogenous PKC activity by phorbol esters strongly inhibits channel
activity (5, 32). These data suggest that
ICl.vol in native cardiac myocytes,
Xenopus oocytes, and recombinant ClC-3 Cl
channels expressed in NIH 3T3 cells are regulated primarily by cPKCs,
in a manner consistent with a model recently proposed which links
changes in cell volume to changes in protein kinase-dependent phosphorylation of the channel (18). Translocation of cPKC
away from the channel during cell swelling would allow the
phosphorylation/dephosphorylation equilibrium of the channel to favor
dephosphorylation and channel opening, whereas translocation of cPKC
toward the vicinity of the channel during cell shrinkage would allow
the phosphorylation/dephosphorylation equilibrium of the channel to
favor phosphorylation and channel closure. This equilibrium would also
be expected to be strongly influenced by the activity of protein
phosphatases, but the possible influence of cell volume changes on
phosphatase activity or distribution remains to be determined. Because
ICl.vol in canine PASMC is also strongly
inhibited by phorbol esters (Fig. 1), it appears that the same general
model might account for cell volume-induced changes in native
ICl.vol in PASMC as well. However, the observed
inability of BIM exposure to activate ICl.vol
under isotonic conditions in these cells suggests the possibility that
different PKC isoforms may be responsible for regulation of
ICl.vol in PASMC.
The possible role of nPKCs in the regulation of
ICl.vol in PASMC was tested using two
PKC-specific inhibitors, calphostin C and the compound Ro-318425, and
two PKC isozyme-specific translocation inhibitory peptides,
V1-2 and
C2-2. Inhibition of endogenous PKC activity by
either calphostin C or Ro-318425 caused activation of native
ICl.vol in PASMC under isotonic conditions and
prevented further modulation of ICl.vol by
anisotonic-induced changes in cell volume. A similar effect was
observed after intracellular dialysis of the
V1-2 peptide, but
not with dialysis of the
C2-2 peptide. These results are
consistent with involvement of nPKC (most likely
PKC), not cPKC
isozymes, in the regulation of ICl.vol in canine
PASMC. Thus dialysis with the inhibitory peptide
V1-2 may
displace endogenous PKC
from its anchoring protein, thus promoting
dephosphorylation and channel activation. Indeed, immunohistochemistry of the subcellular distribution of PKC isozymes in canine PASMC provided evidence that hypotonic cell swelling is accompanied by
translocation of
PKC away from the membrane to cytoplasmic and
perinuclear locations.
Considering the role of protein phosphorylation/dephosphorylation in
the regulation of both native ICl.vol and
recombinant ClC-3 Cl channels proposed here, it is
important to consider the known relationship of PKC to the cytoskeleton
and caveolae, which may be significantly altered during cell volume
changes. An isoform-specific interaction between PKC
and F-actin has
been demonstrated in intact nerve endings, suggesting that F-actin may
be a principal anchoring protein for PKC
(30). An
additional study verified this interaction between PKC
and F-actin
in NIH 3T3 cells and suggested that this protein-protein interaction is
required to maintain PKC
in a catalytically active conformation
(29). Other studies have documented interactions of other
PKC isozymes (PKC
II and PKC
) with F-actin (1, 13).
PKC isozyme-specific interactions with caveolin-1 and -2, scaffolding
proteins that organize a variety of signaling complexes within the
caveolae, have also been demonstrated (26, 31). If F-actin
or caveolin serve as essential anchoring proteins for PKC isoforms,
then a possible mechanism for the activation of
ICl.vol during cell swelling might involve
reorganization of F-actin or alteration or redistribution of caveolar
microdomains during cell swelling (3, 17, 23, 28), causing
PKC translocation to be altered or causing PKC to assume a
catalytically inactive conformation, thus favoring dephosphorylation
and channel activation. Such interactions between PKC and F-actin or
caveolin may also play a role in the ability of some cellular enzymes
and second messengers, known to be altered during cell volume
regulation, to effect changes in ICl.vol,
because PKC isoforms are believed to represent upstream effectors for
several of these pathways as well.
Despite cell-specific differences in PKC isoform regulation of native
ICl.vol in canine PASMC compared with native
ICl.vol in cardiac cells and Xenopus
oocytes, and recombinant ClC-3 channels expressed in NIH 3T3 cells
(5), these currents all exhibit a similar general
sensitivity to regulation by PKC activation and inhibition. In
contrast, native ICl.vol in rabbit portal vein smooth muscle cells exhibits the opposite regulation by PKC, in that
currents are stimulated by activation of endogenous PKC (10, 42). Diversity in the properties of native VSOACs across
different tissues and cell types is well established (27,
34), and future experiments are required to determine whether
differences in PKC regulation reflect different volume-sensitive
intracellular signaling pathways or different molecular entities
responsible for VSOACs in different cell types. The existence of
subtypes of VSOACs may explain the apparent unaltered properties of
native VSOACs in two cell types examined (pancreatic cells and
hepatocytes) from mice with disrupted ClC-3 (Clcn/
knockout) (33).
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Wilson for assistance with the smooth muscle cell dispersion, Drs. D. Duan and B. Horowitz for comments on the manuscript, and Dr. W. A. Large for useful discussions and sharing unpublished data.
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FOOTNOTES |
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This work was supported by National Institutes of Health grants HL-49254 and (National Center for Research Resources) P-20 RR-15581 (to J. R. Hume and I. Yamboliev) and Canadian Institutes of Health Research grant MOP-13101 (to M. P. Walsh). M. P. Walsh is a Medical Scientist of the Alberta Heritage Foundation for Medical Research and a recipient of a Canada Research Chair in Biochemistry.
Present address of J. Zhong: Dept. of Anatomy, Physiology, and Pharmacology, Auburn University College of Veterinary Medicine, Auburn, AL 36849.
Address for reprint requests and other correspondence: J. R. Hume, Center of Biomedical Research Excellence, Dept. of Pharmacology/318, Univ. of Nevada, School of Medicine, Reno, NV 89557-0046 (E-mail: joeh{at}med.unr.edu).
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. Section 1734 solely to indicate this fact.
August 7, 2002;10.1152/ajpcell.00152.2001
Received 23 March 2001; accepted in final form 5 August 2002.
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