Calcium mobilization evoked by hepatocellular swelling is linked to activation of phospholipase Cgamma.

Recovery from swelling of hepatocytes and selected other epithelia is triggered by intracellular Ca(2+) release from the endoplasmic reticulum, which leads to fluid and electrolyte efflux through volume-sensitive K(+) and Cl(-) channels. The aim of this study was to determine the mechanisms responsible for swelling-mediated hepatocellular Ca(2+) mobilization. Swelling of HTC rat hepatoma cells, evoked by exposure to hypotonic medium, elicited transient increases in intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and cytosolic [Ca(2+)]. The latter was attenuated by inhibition of phospholipase C (PLC) with and by IP(3) receptor blockade with 2-aminoethoxydiphenyl borate, but it was unaffected by ryanodine, an inhibitor of intracellular Ca(2+)-induced Ca(2+) release channels. Hypotonic swelling was associated with a transient increase in tyrosine phosphorylation of PLCgamma, with kinetics that paralleled the increases in intracellular IP(3) levels and cytosolic [Ca(2+)]. Confocal imaging of HTC cells exposed to hypotonic medium revealed a swelling-induced association of tyrosine-phosphorylated PLCgamma with the plasma membrane. These findings suggest that activation of PLCgamma by hepatocellular swelling leads to the generation of IP(3) and stimulates discharge of Ca(2+) from the endoplasmic reticulum via activation of IP(3) receptors. By extension, these data support the concept that tyrosine phosphorylation of PLCgamma represents a critical step in adaptive responses to hepatocellular swelling.

Recovery from swelling of hepatocytes and selected other epithelia is triggered by intracellular Ca 2؉ release from the endoplasmic reticulum, which leads to fluid and electrolyte efflux through volume-sensitive K ؉ and Cl ؊ channels. The aim of this study was to determine the mechanisms responsible for swelling-mediated hepatocellular Ca 2؉ mobilization. Swelling of HTC rat hepatoma cells, evoked by exposure to hypotonic medium, elicited transient increases in intracellular levels of inositol 1,4,5-trisphosphate (IP 3 2؉ ]. Confocal imaging of HTC cells exposed to hypotonic medium revealed a swelling-induced association of tyrosine-phosphorylated PLC␥ with the plasma membrane. These findings suggest that activation of PLC␥ by hepatocellular swelling leads to the generation of IP 3 and stimulates discharge of Ca 2؉ from the endoplasmic reticulum via activation of IP 3 receptors. By extension, these data support the concept that tyrosine phosphorylation of PLC␥ represents a critical step in adaptive responses to hepatocellular swelling.

) and cytosolic [Ca 2؉ ]. The latter was attenuated by inhibition of phospholipase C (PLC) with U73122 and by IP 3 receptor blockade with 2-aminoethoxydiphenyl borate, but it was unaffected by ryanodine, an inhibitor of intracellular Ca 2؉ -induced Ca 2؉ release channels. Hypotonic swelling was associated with a transient increase in tyrosine phosphorylation of PLC␥, with kinetics that paralleled the increases in intracellular IP 3 levels and cytosolic [Ca
Hepatocytes undergo dynamic alterations in cell volume in response to changing metabolic demands. For example, under physiological conditions, postprandial uptake of amino acids by hepatocytes results in significant swelling, with subsequent recovery of cell volume (1). Physiological recovery of cell volume requires the opening of plasma membrane K ϩ and Cl Ϫ channels with consequent fluid and electrolyte efflux (2). This process provides a safeguard against irreversible swelling, which, in pathological states, is manifested by ballooning degeneration of hepatocytes (3). Despite the importance of volume-sensitive ion channel regulation in maintenance of normal liver function, the mechanisms that couple hepatocellular swelling to channel activation remain largely undefined.
We have recently shown that K ϩ and Cl Ϫ channel opening evoked by hepatocellular swelling is triggered by an increase in cytosolic [Ca 2ϩ ] ([Ca 2ϩ ] i ) 1 and that this increase is critical for volume recovery (4). The initial increase in [Ca 2ϩ ] i results from Ca 2ϩ mobilization from thapsigargin-sensitive intracellular stores, which suggests the involvement of the endoplasmic reticulum in this process. How Ca 2ϩ is mobilized from the endoplasmic reticulum in response to hepatocellular swelling is as yet unknown.
In extrahepatic tissues, diverse modes of volume-sensitive Ca 2ϩ release from the endoplasmic reticulum exist. In airway epithelium, swelling leads to generation of inositol 1,4,5trisphosphate (IP 3 ) and Ca 2ϩ discharge through IP 3 receptors (5). It would be anticipated that swelling-mediated IP 3 generation is a consequence of cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate by phospholipase C (PLC). In support of a role for PLC, it has been shown in arterial endothelium that inhibition of PLC prevents swelling-induced increases in [Ca 2ϩ ] i (6).
Four major families of PLC isoforms (␤, ␥, ␦, and ⑀) have been recognized, each with distinct modes of activation. In general, PLC␤ is stimulated in response to activation of heterotrimeric G protein-coupled receptors, PLC␥ is stimulated by tyrosine kinases, PLC␦ is stimulated by localized increases in [Ca 2ϩ ] i , and PLC⑀ is stimulated by activation of the monomeric G protein Ras (7). The nature of swelling-activated PLC isoforms has not yet been elucidated.
A second pathway for swelling-mediated Ca 2ϩ mobilization, described in corneal epithelium, involves Ca 2ϩ release through ryanodine receptors (8), although the mechanisms responsible for this remain undefined. A third pathway, reported in vascular smooth muscle cells, involves processes that are independent of both IP 3 and ryanodine receptors (9). However, the underlying mechanisms for swelling-mediated Ca 2ϩ mobilization in these cells have not been further delineated. In hepatocytes, prolonged exposure to hypotonic solution or amino acids (which would tend to increase cell volume) is associated with a small increase in intracellular IP 3 levels (10). Although this raises the possibility that hepatocellular swelling evokes Ca 2ϩ mobilization via activation of IP 3 receptors, it remains to be determined whether this mechanism is involved, and if so, which isoform of PLC is responsible for formation of IP 3 .
In the present study, we have addressed these unresolved issues concerning the origins of volume-sensitive hepatocellular Ca 2ϩ signaling. Here we report in HTC rat hepatoma cells that swelling evoked by hypotonic challenge elicits Ca 2ϩ mobilization by a sequence of events that involve: (a) activation of PLC␥, (b) generation of IP 3 , and (c) activation of IP 3 receptors. We propose that PLC␥ is a pivotal mediator of adaptive responses to increases in liver cell volume and serves a critical role in protection against organ level injury that results from pathological swelling.
Materials and Reagents-Acetoxymethyl esters of fluo-3 and Fura Red were obtained from Molecular Probes. Ryanodine, U73122, and U73343 were purchased from Calbiochem. Monoclonal phosphotyrosine antibody (clone 4G10) and monoclonal paxillin antibody (clone 5H11) were purchased from Upstate Biotechnology, and phosphotyrosine-specific (Tyr-783) PLC␥1 rabbit antibody was from Cell Signaling. Unless otherwise indicated, all other chemicals were obtained from Sigma.
Measurement of IP 3 -IP 3 levels were measured in cultured HTC cells by radioreceptor assay (Amersham Biosciences). Briefly, cells grown on 10-cm culture plates were washed twice with Ca 2ϩ -and Mg 2ϩ -free phosphate-buffered saline (pH 7.4) and then removed with nonenzymatic Cell Stripper (Mediatech). Suspended cells were washed once with SES. Following the wash, cells were centrifuged at 260 ϫ g and then resuspended in SES. Aliquots of 3.6 ϫ 10 6 cells in SES were placed in 1.5-ml centrifuge tubes and incubated in a 37°C water bath for 10 min before swelling with HES. At specific times following hypotonic exposure, cellular IP 3 was extracted by the addition of an equal volume of ice-cold 10% perchloric acid followed by neutralization with an equal volume mixture of freon and tri-n-octylamine (11). Cellular IP 3 was measured according to the manufacturer's directions.

Measurement of [Ca 2ϩ ] i -[Ca 2ϩ
] i was determined in individual cells by dual wavelength microfluorimetry, using the Ca 2ϩ -sensitive fluorescent dyes fluo-3 and Fura Red as described previously (4). Measurements of fluo-3 and Fura Red fluorescence emission intensity were acquired every 10 s, from which the ratio R of fluo-3 to Fura Red fluorescence was calculated. Changes in [Ca 2ϩ ] i were inferred from changes in the relative fluorescence ratio, calculated by dividing R at each time point by R 0 , the mean fluorescence ratio averaged over the initial 2 min of data acquisition. For studies involving inhibitors, comparisons were made with same day controls.
Immunoprecipitation and Immunoblot Analysis-Studies were performed on cells that were plated onto 10-cm dishes and cultured overnight. Prior to hypotonic exposure, cells were equilibrated with SES for 10 min at room temperature. Following this, SES was removed and replaced with either 10 ml of SES (basal) or 10 ml of HES (hypotonic). At designated times, the solutions were replaced with 500 l of ice cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40) plus 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM sodium orthovanadate, and complete protease inhibitor mixture (Roche Molecular Biochemicals). The plates were left on ice for 10 min, and the lysates were harvested. Protein concentration was determined by Bradford reagent (Bio-Rad).
Immunoprecipitations with phosphotyrosine antibody were performed according to the manufacturer's directions, with minor modifications. Phosphotyrosine antibody (3 l) was bound to 50 l of a slurry of washed Protein A-agarose beads by rotating the mixture for 1 h at 4°C. Subsequently, 1 ml of whole cell lysate (1 mg of protein) was added to the beads and gently rocked overnight at 4°C. The beads were washed five times with lysis buffer, resuspended in Laemmli sample buffer, and heated at 100°C for 5 min. The proteins were resolved by SDS electrophoresis on 10% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with PLC␥1 (Tyr-783) antibody, as recommended by the manufacturer. Proteins were detected by exposure to horseradish peroxidase-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratory) and enhanced chemiluminescence (Super Signal, Pierce), and densitometric analysis was performed using an Alpha Imager 2000 (Alpha Innotech).
Immunocytochemistry and Confocal Fluorescence Microscopy-Studies were performed in cells that were seeded onto glass coverslips and cultured overnight. After equilibration with SES for 10 min at room temperature, SES was replaced with either SES (basal) or HES (hypotonic). At designated times, cells were fixed with 4% paraformaldehyde in TBS (Tris-buffered saline, 25 mM, pH 7.4) for 10 min, then perme-abilized for 10 min with 0.2% Triton X-100, 300 mM sucrose in TBS, and gently rocked for 1 h with 1% bovine serum albumin and 10% normal donkey serum (Jackson ImmunoResearch Laboratories). Coverslips were washed twice with TBS and rocked overnight at 4°C with PLC␥1 (Tyr-783) antibody and in selected studies with paxillin antibody. Coverslips were washed three times with TBS and rocked for 1 h at room temperature with Cy3-labeled donkey anti-rabbit Ig F(abЈ) 2 and/or Cy5labeled donkey anti-mouse Ig F(abЈ) 2 (Jackson ImmunoResearch Laboratories). Coverslips were washed three times with TBS and then mounted on slides with Aqua Polymount (Polysciences). Prepared slides were analyzed on a Bio-Rad MRC 1024 ES laser scanning confocal microscope as previously described (12).
Statistics-All results are presented as means Ϯ S.E., where n represents the number of cells studied. Statistical comparisons were made with the use of Student's paired or unpaired t test (as appropriate), and p Ͻ 0.05 was considered to be significant. 3 Levels-We have previously reported that hepatocellular swelling evoked by hypotonic challenge produces a transient increase in [Ca 2ϩ ] i that peaks at 1-2 min and falls to basal values within 10 min (4). In addition, in cultured hepatocytes, prolonged exposure to hypotonic solution causes modest increases in intracellular levels of IP 3 (10). Although these findings raise the possibility that swelling-activated elevations in intracellular IP 3 levels lead to intracellular Ca 2ϩ mobilization, the kinetics of IP 3 generation and the temporal relation to increases in [Ca 2ϩ ] i have not been delineated. It has thus remained unresolved whether IP 3 is a volume-sensitive signaling molecule for intracellular Ca 2ϩ mobilization.

Hepatocellular Swelling Elicits Increases in Intracellular IP
To investigate this further, we determined the time course of IP 3 generation in response to hepatocellular swelling. As shown in Fig. 1, exposure of HTC cells to hypotonic medium elicited a transient increase in intracellular IP 3 levels. The increase in IP 3 levels occurred within 30 s, and these levels approached basal values within 10 min. These kinetics are consistent with the concept that IP 3 elicits intracellular Ca 2ϩ mobilization in response to hepatocellular swelling.
Swelling-evoked Ca 2ϩ Mobilization Requires IP 3 Receptor Activation-The results above suggest that if hepatocellular swelling elicits intracellular Ca 2ϩ mobilization in response to increased intracellular levels of IP 3 , then such Ca 2ϩ increases would be dependent on activation of IP 3 receptors. To test this, we determined the effect of 2-aminoethoxydiphenyl borate (2-APB), an IP 3 receptor inhibitor, on changes in [Ca 2ϩ ] i evoked by hypotonic challenge. As shown in Fig. 2A

. Swelling increases intracellular IP 3 levels in HTC cells.
Cells in suspension were exposed to hypotonic solution (40% reduction in NaCl concentration) at 37°C for the times indicated, and IP 3 was extracted as described under "Experimental Procedures." IP 3 levels were determined by radioreceptor assay (see "Experimental Procedures") and normalized to cell number. Data represent the means Ϯ S.E. of 6 to 9 experiments for each time point.
is possible that the effects of 2-APB were not confined to intracellular Ca 2ϩ mobilization because 2-APB has been suggested to inhibit store-dependent Ca 2ϩ influx in addition to IP 3 receptor blockade (13). To clarify this, we examined, in the nominal absence of extracellular Ca 2ϩ , the effects of 2-APB on swellingmediated changes in [Ca 2ϩ ] i . Under these conditions, contributions to such changes in [Ca 2ϩ ] i that arose from Ca 2ϩ influx would be disabled. In the nominal absence of extracellular Ca 2ϩ , 2-APB ablated increases in [Ca 2ϩ ] i evoked by hypotonic challenge (peak relative fluorescence ratio 1.15 Ϯ 0.06, n ϭ 46 in the presence of 2-APB versus 3.66 Ϯ 0.31, n ϭ 45 in its absence, p Ͻ 0.05). These findings indicate that 2-APB prevented intracellular Ca 2ϩ mobilization elicited by hepatocellular swelling and are consistent with a role for IP 3 receptors in this process.
In addition to IP 3 receptors, it is possible that swellingmediated intracellular Ca 2ϩ mobilization involved ryanodine receptors, as has been reported in corneal epithelial cells (8). We therefore determined the effect of ryanodine, which blocks ryanodine receptors, on changes in [Ca 2ϩ ] i evoked by hypotonic exposure. In contrast to 2-APB, ryanodine did not inhibit swelling-mediated increases in [Ca 2ϩ ] i (Fig. 2B). This observation suggests that ryanodine receptors do not play a role in Ca 2ϩ mobilization evoked by hepatocellular swelling, and it is consistent with the absence of expression of known ryanodine receptor isoforms in hepatocytes (14).
Swelling-evoked Ca 2ϩ Mobilization Requires Activation of PLC-The findings thus far support the concept that hepatocellular swelling elicits intracellular Ca 2ϩ mobilization via generation of IP 3 and activation of IP 3 receptors. It would thus be predicted that swelling-mediated increases in [Ca 2ϩ ] i would require stimulation of phospholipase C, which cleaves IP 3 from phospholipid precursors. To test this, we applied the phospholipase C inhibitor U73122 and determined its effect on changes in [Ca 2ϩ ] i evoked by hypotonic challenge. U73122 prevented such increases in [Ca 2ϩ ] i (Fig. 3). By contrast, U73343, an inactive analog of U73122, did not prevent swelling-mediated [Ca 2ϩ ] i increases (Fig. 3).
Although these findings are consistent with a role for PLC, U73122 has been suggested to inhibit store-dependent Ca 2ϩ influx in addition to inhibition of PLC (15). To minimize contributions arising from Ca 2ϩ influx, we examined, in the nominal absence of extracellular Ca 2ϩ , the effects of U73122 on swelling-mediated changes in [Ca 2ϩ ] i . Under these conditions, U73122 ablated increases in [Ca 2ϩ ] i evoked by hypotonic challenge (peak relative fluorescence ratio 1.08 Ϯ 0.01, n ϭ 69 in the presence of U73122 versus 1.43 Ϯ 0.08, n ϭ 41 in its absence, p Ͻ 0.05). These findings indicate that U73122 prevented intracellular Ca 2ϩ mobilization elicited by hepatocellular swelling and are consistent with a requirement for PLC in this process.
Swelling Elicits Activation of PLC␥-Activation of PLC by cell swelling has been implicated in airway and intestinal epithelium and in vascular endothelium (5,6,16), but neither the mechanisms of activation nor the responsible PLC isoforms have been determined. In liver, selected downstream responses to cell swelling, such as endosomal alkalinization and stimulation of bile flow, have been linked to activation of tyrosine kinases (17,18). We therefore reasoned that PLC␥, an effector of both receptor and non-receptor tyrosine kinase-mediated pathways, could be involved in volume-sensitive hepatocellular Ca 2ϩ signaling. When activated by tyrosine kinases, PLC␥ undergoes tyrosine phosphorylation and becomes associated with the plasma membrane (19,20). With this in mind, we tested whether hepatocellular swelling elicited time-dependent changes in the abundance or localization of tyrosine-phosphorylated PLC␥.
Extracts of HTC cells, taken before and after exposure to hypotonic medium, were analyzed for abundance of tyrosinephosphorylated PLC␥ by immunoprecipitation with anti-phosphotyrosine antibody and subsequent immunoblotting with anti-tyrosine-phosphorylated PLC␥. Increases in tyrosinephosphorylated PLC␥ were evident within 1 min after exposure to hypotonic medium and had fallen to basal values within 10 min (Fig. 4). To localize tyrosine-phosphorylated PLC␥ within the cell following swelling, we performed confocal immunofluorescence microscopy (Fig. 5). As shown in Fig. 5A, at the base of the cells (i.e. within the plane of adherent membrane), under isotonic conditions, tyrosine-phosphorylated PLC␥ was detected in peripheral structures that also labeled for the focal adhesion protein paxillin (21). Localization of tyrosinephosphorylated PLC␥ to focal adhesions is consistent with observations by others (22). Upon exposure to hypotonic medium, the focal adhesion structures labeled by PLC␥ progressively enlarged over the following 8 min, a time at which [Ca 2ϩ ] i was approaching basal values. Midway through the cells, well above the focal adhesion plane, diffuse intracellular staining for tyrosine-phosphorylated PLC␥ could be seen under basal conditions (Fig. 5B). Among cells that stained positively for tyrosine-phosphorylated PLC␥, plasma membrane localization of this protein was evident within 1 min following exposure to hypotonic solution. This would be consistent with a site of action where activated PLC␥ would generate IP 3 from membrane phospholipid substrates. After 1 min, tyrosine-phosphorylated PLC␥ staining in most cells was found to be in a diffuse intracellular pattern, consistent with rapid activated PLC␥ internalization that has been reported by others (19,20). DISCUSSION This study tested whether PLC could act as a trigger for Ca 2ϩ mobilization evoked by hepatocellular swelling. We found that hypotonic swelling of HTC cells elicited a transient increase in IP 3 levels, and swelling-evoked increases in [Ca 2ϩ ] i were attenuated by the PLC inhibitor U73122 and by the IP 3 receptor blocker 2-APB. Moreover, hypotonic exposure led to increased tyrosine phosphorylation of PLC␥ and plasma membrane localization, consistent with PLC␥ activation. The onset of increased PLC␥ phosphorylation paralleled the onset of IP 3 generation, and these events preceded the increase in [Ca 2ϩ ] i evoked by cell swelling. Collectively, these findings support the hypothesis that Ca 2ϩ mobilization evoked by hepatocellular swelling involves a cascade of events that include: (a) activation of PLC␥, (b) production of FIG. 6. Proposed model for hepatocellular volume-sensitive Ca 2؉ signaling. Hepatocellular swelling elicits the activation of a tyrosine kinase, which in turn activates PLC␥. PLC␥ activation leads to generation of IP 3 , which triggers intracellular Ca 2ϩ release from the endoplasmic reticulum (ER) via activation of IP 3 receptors. The resulting increases in [Ca 2ϩ ] i are required for activation of K ϩ and Cl Ϫ channels, which leads to volume recovery.

FIG. 4. Swelling induces tyrosine phosphorylation of PLC␥.
Cells in culture dishes were exposed at room temperature to hypotonic solutions for the times indicated, and cell lysates were generated as described under "Experimental Procedures." Tyrosine-phosphorylated PLC␥ (pY-PLC␥) content was determined by immunoprecipitation with phosphotyrosine antibody and immunoblotting with phosphotyrosine PLC␥1 (Tyr-783) antibody (see "Experimental Procedures"). A, representative immunoblot of immunoprecipitated proteins for pY-PLC␥ under basal conditions and following hypotonic exposure. B, abundance of pY-PLC␥ in HTC cells before and after hypotonic exposure, as determined by densitometric analysis of immunoblots of immunoprecipitated proteins for pY-PLC␥ (see "Experimental Procedures"). Data are normalized to basal values and represent the means Ϯ S.E. of 3 separate experiments. The asterisk indicates p Ͻ 0.05, compared with basal values by paired t test.

FIG. 5. Effects of swelling on the localization of tyrosine-phosphorylated PLC␥ (pY-PLC␥) in HTC cells.
Cells on coverslips were exposed at room temperature to hypotonic solution for the times indicated and fixed as described under "Experimental Procedures." The cellular distribution of pY-PLC␥ by laser confocal fluorescence microscopy of cells stained with phosphotyrosine PLC␥1 (Tyr-783) antibody. A, at the base of the group of cells depicted, pY-PLC␥ (left panels) was found in peripheral structures that co-immunostained with antibody that recognized the focal adhesion protein paxillin (right panels). B, images of cells stained for pY-PLC␥ were taken 4 m above the base. Plasma membrane labeling of pY-PLC␥ can be seen in the cell at the lower right of the panel that corresponds to 1 min after hypotonic exposure. IP 3 , and (c) release of Ca 2ϩ from the endoplasmic reticulum via activation of IP 3 receptors (Fig. 6). Because Ca 2ϩ mobilization is essential for volume recovery after hepatocellular swelling, these data suggest that PLC␥ is a critical mediator of cell volume regulation in liver.
Several important caveats merit comment. First, the compound U73122 has been reported to have effects on [Ca 2ϩ ] i that are independent of inhibition of PLC, in particular inhibition of store-operated Ca 2ϩ influx (15). Second, 2-APB has also been reported to inhibit store-operated Ca 2ϩ influx and thus appears to have effects on [Ca 2ϩ ] i that are independent of IP 3 receptor blockade (13). However, the results of control experiments performed in the absence of extracellular Ca 2ϩ demonstrate that U73122 and 2-APB each inhibited Ca 2ϩ mobilization (and not Ca 2ϩ influx per se) and are thus consistent with blockade of PLC activity and IP 3 receptors, respectively. This interpretation is further supported by two independent assays of PLC activation: measurement of IP 3 levels and tyrosine phosphorylation of PLC␥. It should be noted that although the onset of the increase in tyrosine-phosphorylated PLC␥ paralleled the initial increase in IP 3 levels, it is evident that the kinetics of tyrosine phosphorylation are not exactly identical to those of swellingmediated changes in intracellular levels of IP 3 . The most likely explanation for this is that the IP 3 assays and analysis of tyrosine-phosphorylated PLC␥ were not performed under strictly comparable conditions (e.g. temperature, substrate adherence), and these differences may have influenced the kinetics of PLC␥ phosphorylation and IP 3 generation. Nonetheless, the most parsimonious explanation of our findings is that intracellular Ca 2ϩ mobilization evoked by swelling results from activation of IP 3 receptors in response to PLC␥-mediated production of IP 3 .
Precedent for swelling-mediated activation of PLC exists in airway and intestinal epithelium as well as vascular endothelium (5,6,16). In the latter two tissues, volume-sensitive PLC activation appears to involve autocrine stimulation of P2Y purinergic receptors. Although it is known that P2Y receptors can activate PLC␤ and PLC␥ (23,24), the identity of PLC isoforms that mediate volume-sensitive purinergic Ca 2ϩ signaling has not been elucidated. As we have shown that swelling-mediated increases in [Ca 2ϩ ] i in HTC cells are independent of purinergic receptor stimulation (4), it is likely that distinct mechanisms are involved in volume-sensitive PLC activation in liver. It should be emphasized that we have not excluded a potential role for PLC␤, PLC␦, or PLC⑀ in swelling-evoked [Ca 2ϩ ] i increases, but our findings clearly demonstrate that PLC␥ is tyrosine-phosphorylated upon swelling and thus implicate its involvement in hepatocellular volume regulation. Activation of PLC␥ by swelling would not only account for generation of IP 3 , as we have shown in the present study, but it could be responsible for stimulation (via liberation of diacylglycerol) of protein kinase C␣, which mediates activation of volume-sensitive hepatocellular Cl Ϫ channels (25).
There is good reason to consider PLC␥ as a transducer of signaling events elicited by hepatocellular swelling as several potential upstream activators of PLC␥, including phosphatidylinositol (PI) 3-kinase (26) and tyrosine kinases (7), have been implicated in volume regulatory responses in liver. PI 3-kinase is required for volume recovery after hepatocellular swelling (27), but it is uncertain whether it is involved in swelling-mediated PLC␥ activation. Specifically, activation of PLC␥ by PI 3-kinase appears to be independent of tyrosine phosphorylation (26); yet tyrosine phosphorylation of PLC␥ was seen in the present study. Thus, regulators of PLC␥ other than PI 3-kinase warrant consideration.
Focal adhesion kinase (FAK) undergoes tyrosine phosphoryl-ation upon hepatocellular swelling (28), but its cellular localization in response to swelling has not been delineated. If FAK is involved in volume-sensitive PLC␥ activation, our observations would suggest that focal adhesions may not be the sole site of the swelling-activated interaction between these two proteins because the bulk of plasma membrane-bound tyrosinephosphorylated PLC␥, as detected by immunofluorescence, did not appear to be present in focal adhesions during times corresponding to maximal swelling-induced tyrosine phosphorylation of PLC␥ as determined by immunoblot analysis. Members of the Src family of tyrosine kinases, which can associate with PI 3-kinase (29) as well as FAK (30), mediate volume recovery from swelling in lymphocytes (31) and play important roles in mechanotransduction (32). In lung, mechanical strain elicits Src-mediated PLC␥ activation as a consequence of rearrangements of the actin cytoskeleton (33). Because dynamic actin rearrangements occur in response to hepatocellular swelling (34), this could serve as a mechanism to couple Src to PLC␥ activation in liver. Whether Src kinases, FAK, or PI 3-kinase are involved in volume-sensitive PLC␥ activation remains to be determined.
It is of interest that PLC␥ has been found to be mislocalized or down-regulated in experimental models of liver injury (35,36) that are associated with hepatocellular swelling (37,38). This raises the possibility that PLC␥, through its effects on hepatocellular volume regulation, plays a pivotal role in cytoprotection against pathological swelling. In this way, PLC␥ and its upstream regulators may represent important therapeutic targets for the prevention of organ level injury in pathological conditions associated with disordered control of liver cell volume.