Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC

Juming Zhong1, Ge-Xin Wang1, William J. Hatton1, Ilia A. Yamboliev1, Michael P. Walsh2, and Joseph R. Hume1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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 epsilon V1-2 (10 µM), a translocation inhibitory peptide derived from the V1 region of epsilon PKC, activated ICl.vol under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with beta C2-2 (10 µM), a translocation inhibitory peptide derived from the C2 region of beta PKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of epsilon PKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound epsilon PKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.

chloride channels; cell volume; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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METHODS
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Cell preparation. Mongrel dogs were anesthetized with pentobarbital sodium (45-50 mg kg-1, 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 MOmega . 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(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 80 D-mannitol, and 5 HEPES (pH 7.2, 300 mosmol/kgH2O).

The PKC translocation inhibitory peptides beta C2-2 (MDPNGLSDPYVKL, corresponding to residues 186-198 of beta PKC), epsilon V1-2 (EAVSLKPT, corresponding to residues 14-21 of epsilon PKC), and scrambled epsilon V1-2 (LSETKPAV) were synthesized in the University of Calgary Peptide Synthesis Core Facility using a Beckman model 990B automated peptide synthesizer and purified by preparative reverse-phase HPLC. Phorbol 12,13-dibutyrate (PDBu), calphostin C, Ro-31 8425, and BIM were purchased from Calbiochem (La Jolla, CA). These drugs were first dissolved in dimethylsulfoxide (DMSO) and then further diluted in the solution to a final concentration of DMSO of <0.2%. DMSO alone at 0.2% had no effect on Cl- currents. In Figs. 5-7, the onset of membrane rupture and intracellular dialysis is indicated at time 0.

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-PKCepsilon 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.

For immunofluorescence quantitation, images were acquired of cultured PASMCs subjected to isotonic (n = 11) or hypotonic (n = 10) bath solutions for 20 min, keeping image acquisition parameters constant. Simple PCI software (Compix, Cranberry Township, PA) was used to select regions of interest (ROIs) and calculate pixel intensities. The initial ROIs chosen spanned the entire cell diameter for each image (see Fig. 8, A and C). The pixel intensity across the ROI was then computed, and data were plotted graphically (see Fig. 8, B and D). The ROI was a standard width, with length standardized to account for differences in cell size by having the ROI "overshoot" the membrane on either side of the cell by a fixed amount (2.5 µm). Additional, multiple, smaller ROIs in each cell were sampled in 2 × 4-µm areas juxtaposed either close to the sarcolemmal membrane or close to the nuclear membrane, and mean pixel intensities in these areas were computed to compare differences between these regions in cells exposed to isotonic or hypotonic bath solutions.

Data were expressed as means ± SE (n, no. of cells). Statistical analysis was made by paired t-tests and analysis of variance where appropriate. A value of P < 0.05 was considered to be statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Phorbol 12,13-dibutyrate (PDBu) inhibits hypotonic swelling-induced volume-sensitive Cl- currents (ICl.vol) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Currents were recorded at every 30 s when the membrane potential was stepped to -80 mV () followed by +80 mV (open circle ) from a holding potential of -10 mV. A: time course of currents measured from a cell without any drug application. Inset shows representative currents recorded at the times indicated in A. The cell was superfused with different bath solutions as marked by horizontal bars. B: average current density from a group of cells under different conditions. C: time course of currents measured from a cell exposed to PDBu (100 nM). PDBu was added to the bath solution after the currents reached a steady state in the hypotonic solution. D: average current density from a group of cells exposed to PDBu. Values in B and D represent means ± SE (n = 5 in each group of cells). * Significantly different from the values under isotonic conditions (P < 0.05).

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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Fig. 2.   Lack of effect of bisindolylmaleimide (BIM) on ICl.vol in acutely dispersed canine PASMC. A: time course of currents recorded at -80 mV () followed by +80 mV (open circle ) from a cell that was superfused with isotonic, hypotonic, and hypertonic solutions at times indicated by horizontal bars. BIM was added to the isotonic solution and was removed when the superfusion was switched to a hypotonic solution. B: average current density under different experimental conditions. Values represent means ± SE (n = 7). * Significantly different from the values under isotonic conditions (P < 0.05).



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Fig. 3.   Calphostin C activates ICl.vol in isotonic solutions in acutely dispersed PASMC and prevents further volume regulation of the current. A: representative current recordings when the cell membrane potential was stepped to potentials ranging from -100 to +100 mV in 20-mV increments from a holding potential of -20 mV. a, b, and c show representative current recordings when the cell was superfused with isotonic, isotonic plus calphostin C, and hypotonic solutions, respectively. B: current-voltage relations measured from the same cell under different experimental conditions. C: time course of currents measured at -80 mV () followed by +80 mV (open circle ) from a cell under the same experimental conditions as marked. D: average current density from a group of cells tested under the different experimental conditions. Values represent means ± SE (n = 5). * Significantly different from the values under isotonic conditions (P < 0.05).



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Fig. 4.   Ro-31 8425 activates ICl.vol in isotonic solutions in acutely dispersed PASMC and prevents further volume regulation of the current. A: time course of currents measured at -80 mV () followed by +80 mV (open circle ) from a cell under different experimental conditions as indicated by horizontal bars. B: average current density from a group of cells tested under different experimental conditions. Values represent means ± SE (n = 3). * Significantly different from the values under isotonic conditions (P < 0.05).

These results strongly suggest that, similar to several other cell types, PKC-catalysed phosphorylation and dephosphorylation of PKC-phosphorylated sites play a crucial role in VSOAC regulation by changes in cell volume in canine PASMC. These results also suggest that the isoform of PKC involved in the volume regulation of these channels in PASMC may differ from other cell types. For instance, for native ICl.vol in guinea pig cardiac myocytes, Xenopus oocytes, and recombinant ClC-3 Cl- channels expressed in NIH 3T3 cells, regulation by changes in cell volume may involve cPKCs, because currents were activated under isotonic conditions by exposure to BIM (5, 32), whereas in PASMC regulation of native ICl.vol appears to involve nPKCs.

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. epsilon V1-2 is a short peptide derived from the V1 region of epsilon PKC, which inhibits translocation of epsilon PKC (19, 21). Figure 5 shows the effect of epsilon V1-2 on native ICl.vol in PASMC. Cells were dialyzed with epsilon V1-2 (10 µM) under isotonic conditions and subsequently exposed to hypotonic and then hypertonic solutions. Dialyzing the cells with epsilon 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 epsilon 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 epsilon V1-2 peptide (10 µM), current amplitudes were unchanged under isotonic conditions. In addition, cells dialyzed with scrambled epsilon V1-2 peptide responded in the usual manner to hypotonic and hypertonic solutions (Fig. 5, C and D).


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Fig. 5.   epsilon V1-2 peptide dialysis activates ICl.vol in isotonic solutions in acutely dispersed PASMC and prevents further volume regulation of the current. A: time course of currents recorded at -80 mV () and +80 mV (open circle ) from a cell dialyzed with epsilon V1-2 (10 µM). The cell was superfused with isotonic, hypotonic, and hypertonic solutions as marked with horizontal bars. B: average current densities measured from cells dialyzed with epsilon V1-2 and exposed to different solutions. C: time course of currents recorded at -80 mV () followed by +80 mV (open circle ) from a cell dialyzed with scrambled epsilon V1-2 (10 µM). The cell was superfused with isotonic, hypotonic, and hypertonic solutions as marked with horizontal bars. D: average current densities measured from cells dialyzed with scrambled epsilon V1-2 and exposed to different solutions. Values in B (n = 6) and D (n = 5) represent means ± SE. * Significantly different from the values under isotonic conditions (P < 0.05).

In another test, we examined the effects on ICl.vol in PASMC of beta C2-2, a synthetic peptide derived from the C2 region of beta PKC, which inhibits translocation of conventional (Ca2+-dependent) PKC isozymes (cPKCs) (22). As shown in Fig. 6, intracellular dialysis with beta C2-2 peptide (10 µM) did not evoke any significant change in ICl.vol under basal isotonic conditions, nor did it prevent ICl.vol regulation by cell volume changes. Current densities measured at 1 and 10 min of isotonic superfusion were not different in cells dialyzed with beta C2-2. Subsequent exposure of these cells to hypotonic solutions significantly increased current density, an effect which was reversed by hypertonic superfusion. Taken together, results with PKC translocation inhibitory peptides are consistent with those with different PKC inhibitors and further confirmed that epsilon PKC, a member of the nPKC family, plays a crucial role in cell volume regulation of native ICl.vol in PASMC.


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Fig. 6.   Lack of effect of beta C2-2 peptide dialysis on ICl.vol in acutely dispersed canine PASMC. A: time course of currents recorded at -80 mV () and +80 mV (open circle ) from a cell dialyzed with beta C2-2 (10 µM) under different experimental conditions as marked with horizontal bars. B: averaged current density from a group of cells dialyzed with beta C2-2 and tested under different experimental conditions. Values are means ± SE (n = 5). * Significantly different from the values under isotonic conditions (P < 0.05).

Alterations in cellular distribution of epsilon 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 epsilon 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 epsilon 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. epsilon 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 epsilon 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). epsilon 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 epsilon 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 epsilon PKC from the vicinity of the sarcolemmal membrane to the cytoplasm and perinuclear regions.


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Fig. 7.   epsilon V1-2 peptide dialysis activates ICl.vol in isotonic solutions in cultured canine PASMC and prevents further volume regulation of the current. A-D, same as in Fig. 5. Values in B (n = 4) and D (n = 4) represent means ± SE. * Significantly different from the values under isotonic conditions (P < 0.05).



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Fig. 8.   Digital micrographs showing epsilon PKC-like immunoreactivity (epsilon PKC-LI) in cultured PASMCs exposed to isotonic or hypotonic extracellular solutions. PASMC cells were cultured for 48 h. A: fluorescence digital micrograph showing epsilon PKC-LI in an individual PASMC exposed to isotonic solutions (300 mosmol/kgH2O). C: fluorescence digital micrograph showing epsilon PKC-LI in an individual PASMC exposed to hypotonic (230 mosmol/kgH2O) solution for 20 min. B and D show pixel intensities in selected regions of interest illustrated as boxes in A and C (scanned left to right). Vertical dashed lines indicate cell periphery; horizontal dashed lines estimate level of near-membrane pixel intensity. E: mean pixel intensities for regions of interest in areas juxtaposed close to the sarcolemmal membrane or juxtaposed close to the nuclear membrane (perinuclear) for PASMCs exposed to isotonic (n = 11 cells) or hypotonic bath solutions (n = 10 cells). Values are means ± SE. ** Significantly different from the values under isotonic conditions (P < 0.01). Scale bars, 10 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, epsilon V1-2 and beta 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 epsilon V1-2 peptide, but not with dialysis of the beta C2-2 peptide. These results are consistent with involvement of nPKC (most likely epsilon PKC), not cPKC isozymes, in the regulation of ICl.vol in canine PASMC. Thus dialysis with the inhibitory peptide epsilon V1-2 may displace endogenous PKCepsilon 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 epsilon 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 PKCepsilon and F-actin has been demonstrated in intact nerve endings, suggesting that F-actin may be a principal anchoring protein for PKCepsilon (30). An additional study verified this interaction between PKCepsilon and F-actin in NIH 3T3 cells and suggested that this protein-protein interaction is required to maintain PKCepsilon in a catalytically active conformation (29). Other studies have documented interactions of other PKC isozymes (PKCbeta II and PKCzeta ) 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).


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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