Cell volume regulation in response to hypotonicity is impaired in HeLa cells expressing a protein kinase Calpha mutant lacking kinase activity.

The chloride conductance (G(Cl,swell)) that participates in the regulatory volume decrease process triggered by osmotic swelling in HeLa cells was impaired by removal of extracellular Ca(2+), depletion of intracellular Ca(2+) stores with thapsigargin, or by preloading the cells with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). Furthermore, overnight exposure to the phorbol ester tetradecanoyl phorbol acetate and acute incubation with inhibitors of the conventional protein kinase C (PKC) isoforms bisindolylmaleimide I and Gö6976 inhibited G(Cl,swell). Treatment of HeLa cells with U73122, a phospholipase C inhibitor, also prevented G(Cl,swell). Hypotonicity induced selective PKC alpha accumulation in the membrane/cytoskeleton fraction in fractionation experiments and translocation of a green fluorescent protein-PKC alpha fusion protein to the plasma membrane of transiently transfected HeLa cells. To further explore the role of PKCs in hypotonicity-induced G(Cl,swell), HeLa clones stably expressing either a kinase-dead dominant negative variant of the Ca(2+)-dependent PKC isoform alpha (PKC alpha K386R) or of the atypical PKC isoform zeta (PKCzeta K275W) were generated. G(Cl,swell) was significantly reduced in HeLa cells expressing the dominant negative PKC alpha mutant but remained unaltered in cells expressing dominant negative PKCzeta. These findings strongly implicate PKC alpha as a critical regulatory element that is required for efficient regulatory volume decrease in HeLa cells.

The chloride conductance (G Cl,swell ) that participates in the regulatory volume decrease process triggered by osmotic swelling in HeLa cells was impaired by removal of extracellular Ca 2؉ , depletion of intracellular Ca 2؉ stores with thapsigargin, or by preloading the cells with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N,Ntetraacetic acid). Furthermore, overnight exposure to the phorbol ester tetradecanoyl phorbol acetate and acute incubation with inhibitors of the conventional protein kinase C (PKC) isoforms bisindolylmaleimide I and Gö 6976 inhibited G Cl,swell . Treatment of HeLa cells with U73122, a phospholipase C inhibitor, also prevented G Cl,swell . Hypotonicity induced selective PKC␣ accumulation in the membrane/cytoskeleton fraction in fractionation experiments and translocation of a green fluorescent protein-PKC␣ fusion protein to the plasma membrane of transiently transfected HeLa cells. To further explore the role of PKCs in hypotonicity-induced G Cl,swell , HeLa clones stably expressing either a kinasedead dominant negative variant of the Ca 2؉ -dependent PKC isoform ␣ (PKC␣ K386R) or of the atypical PKC isoform (PKC K275W) were generated. G Cl,swell was significantly reduced in HeLa cells expressing the dominant negative PKC␣ mutant but remained unaltered in cells expressing dominant negative PKC. These findings strongly implicate PKC␣ as a critical regulatory element that is required for efficient regulatory volume decrease in HeLa cells.
Cells undergo dynamic changes in volume in response to different stimuli, including variations in extracellular tonicity. Upon cell swelling by exposure to hypotonic conditions, transport pathways for organic osmolytes as well as K ϩ and Cl Ϫ channels are activated and accompanied by movement of water to the external medium in a process known as regulatory volume decrease (RVD) 1 (1,2). In mammalian tissues most of the cells are exposed to extracellular fluid with well controlled osmolarity. A remarkable exception is the kidney medulla, where extracellular osmolarity exceeds isotonicity by Ͼ4-fold. Less dramatic changes of extracellular osmolarity occur during intestinal absorption, which exposes intestinal cells to anisosmotic luminal fluid. Even at constant extracellular osmolarity cell volume is compromised by alterations of intracellular osmolarity. Several metabolic pathways lead to cellular formation or degradation of osmotically active substances. Furthermore, transport across the cell membrane modifies cellular osmolarity and, thus, cell volume (for review, see Ref. 2).
In addition, cell volume changes have important physiological roles as integral components of the signal transduction events involved in diverse processes such as proliferation, apoptosis, migration, exocytosis, and neuronal excitability (3,4). Under pathological conditions cell swelling has been also observed, namely during correction of extracellular acidosis in the course of the treatment of diabetic ketoacidosis (5). Although the precise mediators and pathways that connect cell swelling to the activation of G Cl,swell and, thus, RVD remain largely unidentified. Possible mechanisms involved include cytosolic changes in ionic strength, macromolecular crowding (6,7), and variations in intracellular Ca 2ϩ concentration (8). In addition to Ca 2ϩ other intracellular components, such as ATP (9 -11), have been implicated as mediators of G Cl,swell in certain cell types.
Protein phosphorylation also plays a role in the modulation of G Cl,swell . Activation of G Cl,swell by tyrosine kinases has been shown to occur in some cells, such as cardiac myocytes (12), H4IIE hepatoma cells (13), and I407 cells (14). In various cell types protein kinase Cs (PKCs) appear involved in modulating G Cl,swell (15)(16)(17). In Ehrlich ascites tumor cells PKCs were found to be activated in the later phases of RVD, and it was suggested that PKCs may play a role in terminating the RVD response, presumably by modulating the Na ϩ -K ϩ -2Cl Ϫ cotransporter (18).
The PKC family includes at least 11 members that are generally classified in three categories based on cofactor requirements for activation and sequence homologies. Conventional Ca 2ϩ -dependent PKCs (cPKCs) are activated by DAG in the presence of Ca 2ϩ , whereas novel PKCs are Ca 2ϩ -independent. Both groups are, however, activated by phorbol esters. In contrast, atypical PKCs are neither activated by DAG nor by phorbol esters (19 -24).
HeLa cells have been extensively used as a model to study RVD (25)(26)(27) and, thus, are well suited to address questions concerning the role of PKCs and the signal transduction cascades involved in G Cl,swell activation. Here, we examined the effect of PKCs on G Cl,swell by monitoring in intact cells the time course of activation of the volume-activated Cl Ϫ conductance.
Because conditions under which patch clamp whole-cell recordings are carried out are significantly different from intact cell experiments, we have pursued this question using an intactcell technique (27). Experiments described here established the importance of both extracellular and intracellular Ca 2ϩ for G Cl,swell activation in HeLa cells. In addition, PKCs were implicated by pharmacological manipulation with inhibitors, and a role for PKC␣ was inferred from translocation experiments.
To specifically determine the contribution of PKC␣ to G Cl,swell activation, HeLa cell lines stably expressing either dominant negative PKC␣ (K386R) or PKC (K275W) were generated and studied. Hypotonicity-induced G Cl,swell was found to be severely impaired in all lines expressing dominant negative PKC␣ (K386R), whereas the presence of PKC (K275W) had no effect. These results implicate PKC␣ as an important regulatory element controlling RVD in HeLa cells.
Western Blot Analysis-The presence of the different wild type PKC isoforms and expression of the dominant negative PKC␣ (K386R) and PKC (K275W) mutants was determined by Western blot analysis using either PKC isoform-specific or His 6 -tag-specific antibodies, respectively. Cells were lysed and sonicated in a minimal volume of lysis buffer (containing 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 10 g/ml leupeptin, 10 g/ml trasylol, 1 mM phenylmethylsulfonyl fluoride, and 50 mM Hepes, pH 7.5) and centrifuged. Protein content of cell lysates was determined by the Bradford method (28). Proteins in whole-cell lysates were dissolved in sample buffer (29), separated by SDS-PAGE in 8% gels, and subsequently transferred to nitrocellulose as described (30). Nonspecific binding sites of the membrane were blocked using 5% nonfat milk in 0.05 M Trisbuffered saline, pH 7.2. PKC isoforms in HeLa cells were identified using different dilutions of mouse monoclonal antibodies against PKC ␣, ␦, ⑀ (Transduction Laboratories, KY) or rabbit polyclonal antibodies against PKC ␤ 2 , , , and (Santa Cruz Biotechnology, Santa Cruz, CA), as previously described (31). A mouse polyclonal antibody against a His 6 -tagged salmon fusion protein (BiosChile, Santiago, Chile) was used to identify expression of PKC mutant isoforms in transfected HeLa cells. Non-specifically bound antibody was removed by washing with Tris-buffered saline containing 0.1% Tween 20. Specific PKC and His 6 reactivity were determined using either peroxidase-conjugated goatanti mouse or goat-anti-rabbit IgG antibodies (Jackson Immuno-Research Laboratories). The peroxidase activity was detected using the ECL chemiluminescence reagent (PerkinElmer Life Sciences) and revealed by exposure to x-ray films (Eastman Kodak Co.). Immunoreactive bands present on the films were scanned. Intensity values were obtained after scanning densitometry of individual bands with Scion-Image (NIH) software.
Down-regulation of PKC-In PKC down-regulation studies cells were incubated for 24 h with 1 M TPA in the culture medium. Control cells were incubated with the vehicle Me 2 SO (0.1%) for 24 h.
Analysis of PKC Translocation by Cell Fractionation-Cells were homogenized by pipetting 30 times through a 25-gauge needle and centrifuged at 280 ϫ g for 10 min to eliminate nuclei. The supernatant was then centrifuged at 100000 ϫ g for 1 h. The resulting soluble supernatant (cytosolic) fraction was stored at Ϫ80°C, whereas the residual pellet was resuspended in the same buffer supplemented with 1% Triton X-100, sonicated for 1 min, and stored at Ϫ80°C (particulate fraction). Before Western blot analysis the protein concentration was determined using the Bradford assay. Equal amounts of protein (100 g) were loaded per lane and separated by SDS-PAGE as described above.
In Situ Analysis of PKC Translocation Using GFP-tagged Proteins-HeLa cells were plated on glass bottom dishes (MatTek Corp., Ashland, MA) 1 day before liposome-mediated transfection with LipofectAMINE 2000 according to the manufacturer's instructions (Invitrogen) of the PKC␣-GFP or PKC-GFP construct (BD Biosciences and Clontech, Palo Alto, CA, respectively). Half a microgram of each plasmid and 3 l of LipofectAMINE 2000 were used for 5.5 ϫ 10 4 cells. After 4 h in the presence of LipofectAMINE, cells were transferred to normal medium for 24 -28 h. Cells were then washed with phosphate-buffered saline and maintained in isotonic solution. The fluorescence-imaging experiments in live cells were performed at room temperature (20 -25°C). Images were obtained using a laser-scanning confocal system coupled to a 510 Zeiss microscope with an immersion objective (40ϫ, 1.2 numerical aperture) and subsequently analyzed with the software ImageJ, version 1.30p (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, MD, rsb.info.nih.gov/ij) to determine relative PKC-GFP intensity distribution. Cells were imaged either under isotonic conditions (control) after exposure to 30% hypotonicity or after TPA treatment.
Construction of HeLa Cell Lines Stably Expressing either PKC␣ or PKC Dominant Negative Mutants-HeLa cells were stably transfected with a bovine PKC␣ Lys/Arg 368 and PKC Lys/Trp 275 cDNA mutant containing a His 6 tag at the COOH terminus subcloned into the cytomegalovirus expression vector pEF-neo (32). Cells were transfected using LipofectAMINE as indicated by the manufacturer (Invitrogen), and clones were selected with 200 g/ml G418 in the culture medium.
Cell Volume Measurements-Changes in cell water volume of individual cells were assessed by measuring variations in the concentration of an intracellularly trapped fluorescent dye as previously described (27,33). Briefly, HeLa cells were loaded with 5 M calcein-AM (Molecular Probes, Eugene, OR) for 5 min and then superfused with an iso-osmotic solution for 15 min before subjecting the cells to hypotonicity. Changes in fluorescence were monitored using a Zeiss LSM 410 confocal microscope as previously described. Images were obtained at 10-s intervals, and the fluorescence of a 10-m 2 area in the center of a cell was measured. The data are presented as V t /V 0 values, where V 0 is the cell water volume in iso-osmotic solution at time 0, and V t is the cell water volume at time t. V t /V 0 was calculated from the fluorescence intensity ratio F 0 /F t as described (33).
Cell Calcium Measurements-Relative changes in intracellular Ca 2ϩ were assessed by measuring variations in the fluorescence of the Ca 2ϩsensitive dye Rhod-2 (Molecular Probes) (34). Cells attached to glass coverslips were loaded for 20 -40 min at 37°C with saline containing the acetoxymethyl ester of the dye (Rhod-2AM, final concentration, 5.2 M and 0.1% pluronic acid). Coverslips were placed at the bottom of a 1-ml capacity chamber and submerged initially in an iso-osmotic solution. Fluorescence changes at emission wavelength 570 nm, as a consequence of exposure to hypotonic conditions, were measured using a Zeiss LSM 410 confocal microscope and an excitation wavelength of 543 nm. Images were scanned every 10 s. The images were analyzed off-line with ImageJ, version 1.30p. The relative fluorescence intensity ratio (F t /F 0 ) is expressed as a function of time.
Statistical Analysis-Statistical differences of the data were evaluated by unpaired Student's t test and considered significant at p Ͻ 0.05.
Reagents and Treatments-The phorbol ester TPA (Sigma), Gö6976, BIM-1, U-73122, BAPTA-AM (all from Calbiochem) were dissolved in Me 2 SO (final Me 2 SO concentration never exceeded 0.1% in the culture medium) and stored at Ϫ20°C for up to 1 month. In all cases cells were preincubated in the presence of the different PKC modulators (20 nM TPA, 500 nM BIM-1, or 100 nM Gö6976), the PLC inhibitor U73122 (1 M), the IP 3 receptor blocker 2-aminoethoxydiphenil borate (50 M), and thapsigargin (1 M) for 30 min. Cells were then exposed to hypotonicity, and volume or Ca 2ϩ changes were measured. Inhibitors were purchased from Calbiochem and Sigma. All other reagents were of analytical grade and were purchased from Sigma, Aldrich, or Merck.

Expression of Protein Kinase C Isoforms in HeLa Cells-PKC
isozymes expressed in HeLa cells were detected as bands of the predicted molecular weights by Western blot analysis of whole cell lysates using isotype-specific antibodies against PKC␣, -␤ 2, -␦, -⑀, -, and - (Fig. 1). Bands observed in HeLa cell extracts comigrated with the corresponding bands detected in tissue extracts employed as controls for each PKC isotype (brain for -␣, -␤ 2 , -␦, and -; skeletal muscle for -⑀, -, and -). The isozyme PKC was not detected in HeLa cell lysates under the experimental conditions used. These results indicate that at least one isoform of each PKC subgroup (cPKCs, novel PKCs, and atypical PKCs) was expressed in HeLa cells.
G Cl,swell in HeLa Cells Is Ca 2ϩ -dependent and Blocked by TPA-Calcium has been shown to be an intracellular mediator or potentiator of hypotonicity-induced G Cl,swell in some cells, in particular epithelial cells (1,3). Thus, the effect of Ca 2ϩ on G Cl,swell was explored next. Under control conditions, cells rapidly swelled after exposure to a 30% hypotonic NaCl solution (Fig. 2). G Cl,swell was monitored by switching to a hypotonic NMDG-Cl solution containing 10 M of the cation-selective ionophore gramicidin (27). The rationale of this experimental procedure is to make Cl Ϫ conductance rate-limiting for net ion transport across the plasma membrane. This is achieved by replacement of all monovalent cations in the medium by the impermeant cation NMDG and the addition of gramicidin. Under these conditions the conductance of the membrane is dominated by the K ϩ permeability and, should a Cl Ϫ conductance be present, the cell will shrink at a rate proportional to anion permeability (27). Upon exposure to a 30% hypotonic solution the cells immediately swelled, indicating that cell volume was adequately monitored in these experiments. Under these experimental conditions, cells typically displayed excessive volume recovery (control trace in Fig. 2), a fact that may be explained by the absence of an appropriate ion gradient for the Na ϩ -K ϩ -2Cl Ϫ transporter in Na ϩ -free medium. HeLa cells were preloaded with 100 M BAPTA-AM to deplete bulk cytoplasmic free Ca 2ϩ . Under these conditions G Cl,swell was markedly retarded as compared with control cells (Fig. 2 and Table  I). Different sets of experiments were conducted to identify the origin of Ca 2ϩ . The participation of extracellular Ca 2ϩ in G Cl,swell was studied by removal of the divalent cation from the medium. As depicted (Fig. 2), this treatment impaired G Cl,swell to a similar degree as seen in the BAPTA-loaded cells. The role of intracellular Ca 2ϩ was addressed by long term preincubation of cells with 1 M thapsigargin in the presence or absence of extracellular Ca 2ϩ . These conditions (Table I) reduced G Cl,swell to a similar extent as seen when Ca 2ϩ was omitted from the medium, indicating that Ca 2ϩ of intra-and extracellular origin regulate the rate of hypotonicity-induced G Cl,swell in HeLa cells.

Regulatory Volume Decrease in HeLa Cells Is Modulated by PKC␣
The results described above prompted us to examine intracellular Ca 2ϩ changes induced by hypotonicity under the different experimental conditions. A 30% decrease in extracellular hypotonicity in a Ca 2ϩ -containing hypo-osmotic medium triggered a biphasic Ca 2ϩ response. The rapid more transient component terminated within 5-6 min, whereas the slower sustained component was maintained up to 10 -12 min. Upon removal of extracellular Ca 2ϩ , only the faster transient component was retained but displayed slightly slower kinetics and a smaller amplitude. After a 30-min exposure to 1 M thapsigargin in the presence or absence of extracellular Ca 2ϩ , hypotonicity failed to produce any significant variation in intracellular Ca 2ϩ (Fig. 3). Peak fluorescence intensity of the rapid component in the presence of extracellular Ca 2ϩ was 1.54 Ϯ 0.02, compared with 1.31 Ϯ 0.01 in the absence of Ca 2ϩ . Fluorescence intensity for the slower component was 1.28 Ϯ 0.05 in the presence of extracellular Ca 2ϩ compared with 1.01 Ϯ 0.02 in the absence of Ca 2ϩ (inset in Fig. 3).
Long term incubation with TPA has been shown previously to down-regulate cPKC and novel PKC protein levels by promoting proteolytic degradation. This often occurs at different rates for individual isoforms (19). Therefore, we investigated the effect of overnight exposure of HeLa cells to 1 M TPA on G Cl,swell . As suspected, cell volume recovery was significantly delayed in TPA-treated cells upon hypotonic challenge ( Fig. 2 and Table I), suggesting that TPA-sensitive PKC(s) regulated G Cl,swell in HeLa cells. Western blot analysis of HeLa cells after overnight incubation with TPA revealed a significant decrease in PKC␣ protein levels. Scanning densitometry analysis of PKC␣ before and after TPA treatment confirmed that PKC␣ levels were reduced by at least 50%, whereas PKC levels remained essentially unaltered (Fig. 4, A and B).
Hypotonicity Induced Translocation of PKC␣ to the Particulate Fraction of HeLa Cells-PKC activation is often associated with PKC translocation to the plasma membrane. To investigate this possibility cells were initially fractionated as described, and the presence of PKCs in the particulate (mem-brane) fraction was assayed by Western blotting. Brief (5 min) exposure of HeLa cells to hypotonic conditions resulted in an increase by 47.5 Ϯ 4.7% of PKC␣ in the particulate fraction, whereas no changes were observed for PKC (Fig. 5, A and B).
Hypotonicity Induced Translocation of GFP-PKC␣ to the Plasma Membrane of HeLa Cells-The results above demonstrated that PKC␣ specifically altered its intracellular distribution in response to hypoosmotic cell swelling and accumulated in a preparation that included the plasma membrane. To implicate PKC␣ more directly in a model for RVD control in HeLa cells, we anticipated that PKC␣ should translocate to the plasma membrane to induce changes in G Cl,swell activity. To test this model translocation of GFP-tagged wild-type PKC␣ and PKC were compared in transiently transfected HeLa cells. GFP-PKC␣-or GFP-PKC-transfected cells maintained in either isotonic medium or after exposure to 30% hypotonicity for 4 min were examined by laser-scanning confocal microscopy (Fig. 6, A and B, respectively). In Fig. 6, A and B, a representative experiment is depicted showing cells for which dimensions in the x,y plane were conserved upon exposure to hypotonicity. Hence, swelling of these cells occurs along the z axis only. Cells in which dimensions in the x,y plane were not conserved upon swelling are also shown (Fig. 6A, inset). Only cells in which swelling occurred along the z axis were chosen for statistical analysis. This analysis was performed by taking a section across the cell that included the portion of the membrane showing fluorescence in iso-and hypotonicity. Then, the area under the fluorescence intensity profile corresponding to the membrane in isotonicity was subtracted from the profile obtained in hypotonicity. GFP-PKC␣ fluorescence increased from 1.04 Ϯ 0.07 to 1.32 Ϯ 0.09 (p Ͻ 0.05, n ϭ 12) in hypotonicity, whereas GFP-PKC fluorescence changed from 1.01 Ϯ 0.09 to 1.11 Ϯ 0.06 (p Ͼ 0.05, n ϭ 12) under the same conditions. The differences in relative fluorescence intensity (⌬), calculated by subtracting the fluorescence detected in hypotonicity minus isotonicity, is depicted (bottom panels, Fig. 6, A  and B). In the case of GFP-PKC␣, arrows indicate fluorescence spots associated with plasma membrane regions in which the relative fluorescence intensity was higher after hypotonic stimulus and, thus, indicative of translocation to the plasma membrane. In the case of GFP-PKC no difference was observed. As an additional control, cells transiently transfected with GFP-PKC␣ were exposed to 20 nM TPA (Fig. 6C) for 4 min in isotonicity. The difference in relative fluorescence intensity indicated significant translocation of GFP-PKC␣ to the plasma membrane. As expected, TPA treatment of GFP-PKC-expressing HeLa cells did not promote translocation of GFP-PKC (data not shown). Quantitative analysis of such translocation experiments indicated that PKC␣ but not PKC accumulated in plasma membrane proximity under hypotonic conditions (Fig. 6D).
Pharmacological Inhibition of PKC, PLC, or the Endoplasmic Reticulum Ca-ATPase Impaired G Cl,swell -To implicate further PKCs and specifically PKC␣ in the control of RVD, HeLa cells were treated with BIM-1 and Gö6976, two compounds known to be fairly selective inhibitors for cPKC isoforms when used at appropriate concentrations (35). As depicted in Table I, G Cl,swell -elicited (by 30%) extracellular hypotonicity was significantly impaired when cells were pretreated with either BIM-1 or Gö6976, whereas maximal V t /V 0 was comparable in inhibitor and Me 2 SO-treated cells. Given that both BIM-1 and Gö6976 are PKC kinase domain inhibitors with some preference for the PKC␣ isoform (35), these results further implicated PKC␣ in controlling G Cl,swell in HeLa cells.
DAG, in conjunction with Ca 2ϩ , is a physiologically relevant activator of cPKCs in numerous cellular systems (21). The results presented so far supported the notion that RVD in HeLa cells was dependent on increased intracellular Ca 2ϩ , partially generated by intracellular Ca 2ϩ mobilization, perhaps via an IP 3 -dependent mechanism. To test this, HeLa cells were treated with the IP 3 receptor blocker 2-aminoethoxydiphenil borate. This treatment significantly impaired G Cl,swell (Table I), thus implicating further this second messenger in the signaling pathways controlling G Cl,swell . In addition, inhibition of the endoplasmic reticulum Ca-ATPase with thapsigargin significantly blunted G Cl,swell (Table I) presence of U73122 (1 M) and absence of extracellular Ca 2ϩ before hypotonic challenge. Immediately after hypotonic exposure, U73122-treated and control cells rapidly increased their volume to values observed in nontreated cells (compare Fig. 2 and Table I). However, in U73122-treated cells G Cl,swell was substantially slower (Table I). These results indicate that a PLC isoform participates in the intracellular signaling cascade leading to RVD in HeLa cells and possibly activates cPKC␣ via DAG and IP 3 -dependent calcium release.
Expression of Dominant Negative PKC␣ and PKC Mutants-Pharmacological inhibitors are important tools for implicating signal transduction effector proteins in specific cellular processes. However, because such inhibitors have additional, often unknown, intracellular targets, HeLa cell lines stably expressing either a dominant negative PKC␣ (K368R) or PKC (K275W) mutant were generated. Five different PKC␣ (K368R)-and four different PKC (K275W)-expressing HeLa clones were characterized and yielded similar results. The immunoreactivity pattern of PKC␣ and PKC from representative HeLa clones overexpressing the PKC isoforms is shown. Scanning densitometric analysis revealed a 4.6-fold increase in the expression of PKC␣ (K386R) and a 3-fold increase for PKC (K275W) (Fig. 7A). As expected, PKC␣ and PKC were detected in homogenates of parental HeLa cells and transfected clonal populations (Fig. 7A). In addition, a band of the same size as the corresponding PKC was detected using a His 6 -specific antibody in PKC␣ (K368R)-and PKC (K275W)-transfected cells, whereas that was not the case for parental (wt) and mock-transfected (mock) HeLa cells (Fig. 7B).
Hypotonicity-induced G Cl,swell Is Impaired in HeLa Cells Expressing the Dominant Negative PKC␣ Mutant-Hypotonicityinduced G Cl,swell response was studied using these stably transfected cell lines expressing either PKC␣ (K386R) or PKC (K275W). RVD was significantly impaired in PKC␣ (K386R) HeLa cells as compared with PKC (K275W) transfected cells ( Fig. 8 and Table I). These results demonstrate that cPKC␣ is a critical component in signaling mechanisms that control G Cl,swell in HeLa cells. DISCUSSION Volume regulation is considered a crucial physiological mechanism required for adequate cell functioning. RVD subsequent to cell swelling occurs due to the activation of channels and transporters that allow the efflux of KCl and organic osmolytes. The mechanisms that regulate these processes are complex and differ among various cell types (36). For instance, extracellular hypotonicity has been shown to mobilize intracellular Ca 2ϩ that was partially required to sustain K ϩ efflux in astrocytes (37). Also, PKC-dependent activation of volume-sensitive chloride channels has been suggested to occur in HT-3 cells (16,17). In general, however, the processes that modulate cell volume recovery are not well understood. Here, using HeLa cells as a model system, intra-and extracellular Ca 2ϩ mobilization, PLC activation, and most importantly, unequivocal evidence implicating PKC␣ acti- vation in the sequence of events controlling cell volume recovery after osmotic swelling are provided.
A significant decrease in G Cl,swell was observed after exposure to Ca 2ϩ -free hypotonic medium (Fig. 2). This observation indicates that hypotonicity may activate Ca 2ϩ influx through different membrane pathways, including stretch-activated Ca 2ϩ channels (38,39). Moreover, it has been shown that several types of cells undergo RVD only when Ca 2ϩ influx from the extracellular compartment takes place (8).
Suppression of swelling-associated intracellular Ca 2ϩ increases with BAPTA-AM significantly reduced G Cl,swell in HeLa cells as did also thapsigargin and 2-aminoethoxydiphenil borate treatment (Fig. 2 and Table I), suggesting that Ca 2ϩ release from intracellular stores via an IP 3 -dependent mecha-nism is critical for volume recovery in these cells. This conclusion is in agreement with results obtained in astrocytes where Ca 2ϩ mobilized from the endoplasmic reticulum was suggested to be the source of Ca 2ϩ required for hypotonicity-stimulated K ϩ release (37,40). In addition, swelling has been associated in a wide variety of cells with intracellular Ca 2ϩ increases that activate K ϩ and Cl Ϫ efflux via channels, loss of water, and as a consequence, recovery of cell volume (8,41). Nonetheless, this mechanism is not generic, even among epithelial cell lines. For example, the volume-activated K ϩ current in Ehrlich ascites tumor cells is activated even under conditions where intracellular Ca 2ϩ is buffered (42).
The results obtained by measuring relative intracellular Ca 2ϩ variations after cell exposure to hypotonicity (Fig. 3) coincide well with the findings showing Ca 2ϩ dependence of G Cl,swell in HeLa cells (Fig. 2). In addition, they underscore the importance of Ca 2ϩ mobilization from thapsigargin-sensitive intracellular stores in generating the Ca 2ϩ signal responsible for G Cl,swell . After long term (30 min) exposure to thapsigargin, intracellular Ca 2ϩ variations in response to hypotonicity were not detectable even in the presence of extracellular Ca 2ϩ (Fig.  3). Similar results to those described here have been obtained in renal A6 cells (43). Depletion of internal Ca 2ϩ stores by acute treatment with thapsigargin would be expected to trigger Ca 2ϩ influx via store-activated plasma membrane channels and, hence, augment cytosolic Ca 2ϩ observed in response to hypotonicity. However, in experiments conducted here cells were preincubated for 30 min with thapsigargin. Clearly, this protocol depleted intracellular stores such that Ca 2ϩ transients were no longer detectable upon exposure to hypotonicity (Fig. 3), and as a consequence, G Cl,swell was blocked ( Table I).
Because of the above findings the participation of phospholipases was investigated. Indeed, marked reduction of G Cl,swell was observed with the PLC inhibitor U73122 in the absence of extracellular Ca 2ϩ (Table I). A role for PLCs in the regulation of osmo-sensitive K ϩ release has been reported (44), and calcium mobilization triggered by hepatocellular swelling was more recently associated with PLC␥ activation via tyrosine kinases (41). Activation of tyrosine kinases during RVD has been demonstrated in cardiac myocytes (12), H4IIE hepatoma cells (13), and I407 cells (14), and in Ehrlich ascites tumor cells the tyrosine kinase inhibitor genistein inhibits the RVD response (3). Consistent with the notion that tyrosine phosphorylation plays a critical role in RVD in HeLa cells, possibly via Extracts were prepared from stably transfected cell lines as described, and 50 g of total protein from parental (wt) and mock-and PKCtransfected cells were analyzed. A, representative immunoblots of parental (wt) and mock and dominant negative His 6 -tagged PKC proteins were revealed using a specific monoclonal antibody against PKC␣ and a polyclonal antibody against PKC. B, the same samples were revealed using an anti-His 6 antibody. His 6 -tagged PKC␣ (K386R) or PKC (K275W) protein bands (82 and 72 kDa, respectively) were detected exclusively in the transfected clones. A 34-kDa His 6 -tagged salmon fusion protein (control) was used as a positive control for the His 6 antibody. Molecular weights are indicated to the left of individual panels.  Table I. PLC activation, the Src-family kinase inhibitor PP-1 was also found in preliminary experiments to block swelling-induced taurine efflux (53).
The role of serine/threonine protein kinases in the regulation of cell volume recovery is less well understood, although they have been implicated in the late phase of RVD in Ehrlich ascites tumor cells (18). Also, a tight connection between calmodulin activation and organic osmolyte fluxes evoked by hypotonicity has been reported in some epithelial cells (45,46). Furthermore, evidence suggesting a role for PKC in the activation of the swelling-dependent Cl Ϫ conductance was provided in HTC hepatoma and Mz-ChA-1 cells (17). In those experiments reagents such as chelerythrine, calphostin C, and phorbol esters, all currently known to display modest selectivity for PKCs (47)(48)(49)(50)(51), were employed. Also, upon microinjection of HT-3 cells with anti-PKC␣ antibodies at high concentrations, RVD was partially blocked at times greater than 10 min, and the time course of RVD was prolonged (16). Finally, chelerythrine, a highly controversial PKC inhibitor, as mentioned above (50,51), was found to decrease the RVD response in Ehrlich ascites tumor cells (3). These experiments all provided suggestive, but not conclusive, evidence that PKCs were involved in events leading to G Cl,swell activation.
Hence, we were interested in investigating the importance of PKCs as potential downstream effectors required for G Cl,swell activation in HeLa cells as well as in identifying unequivocally the PKC isoform involved. Indeed, initial experiments with BIM-1 and Gö6976, compounds known to be relatively selective cPKC inhibitors, blocked G Cl,swell very efficiently (Table I).
Although pharmacological inhibitors are valuable experimental tools in helping to identify relevant cell signaling pathways and proteins, most are known to have multiple intracellular targets, as has been discussed for chelerythrine, calphostin C, and phorbol esters. To implicate further PKCs and, specifically, cPKC␣ in the control of the swelling-activated Cl Ϫ conductance and, hence, RVD in HeLa cells, additional experimental approaches were employed. Experiments analyzing translocation of PKC␣ in response to hypotonicity indicated that this isoform altered its distribution and accumulated in the particulate fraction (Fig. 5), as has been described previously in HTC hepatoma cells (17). No such changes were seen for PKC employed as a control (Fig. 5). Furthermore, selective translocation of GFP-tagged PKC␣, but not PKC, to the plasma membrane upon exposure of HeLa cells to hypotonicity was observed. Thus, our experiments demonstrate that in response to hypotonicity PKC␣ translocates to the subcellular site (i.e. the plasma membrane), where its activation is expected to control the swelling-sensitive Cl Ϫ conductance.
Direct confirmation that PKC␣ is critically involved in controlling G Cl,swell in HeLa cells was obtained using HeLa cells lines stably expressing either dominant negative PKC␣ (K368R) or PKC (K275W). These mutations in the ATP binding site preclude catalytic activity of the respective isoforms (32). Only in the case of PKC␣ (K368R) was G Cl,swell significantly delayed (Fig. 8). The control using a corresponding PKC mutant had no consequence on G Cl,swell , thereby eliminating the possibility of a promiscuous effect, for instance, due to sequestration of upstream kinases such as PDK1, essential for activation of several PKCs (52). It should be noted that the PKC mutant displayed slower G Cl,swell activation in comparison to non-transfected cells (see Fig. 2). Although these observations suggest that the transfection/selection procedure per se altered G Cl,swell activation in HeLa cells, an additional effect due to the presence of PKC␣ (K386R), but not with PKC (K275W), was clearly distinguishable.
In conclusion, the data presented support a model in which hypotonicity triggers extracellular Ca 2ϩ influx, PLC activation, and intracellular Ca 2ϩ mobilization. These events are crucial to the onset of the swelling-activated Cl Ϫ conductance and, thus, RVD in HeLa cells. Downstream of this initial sequence, translocation of cPKC␣ to the plasma membrane occurs, and activity of this isoform is required to mediate some of the processes leading to hypotonicity-induced cell volume regulation in HeLa cells.