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Originally published In Press as doi:10.1074/jbc.M301958200 on August 25, 2003

J. Biol. Chem., Vol. 278, Issue 45, 44632-44638, November 7, 2003
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Volume-sensitive Tyrosine Kinases Regulate Liver Cell Volume through Effects on Vesicular Trafficking and Membrane Na+ Permeability*

Andrew P. Feranchak{ddagger}, Gordan Kilic§, Paul A. Wojtaszek§, Ishtiaq Qadri§, and J. Gregory Fitz§

From the Departments of {ddagger}Pediatrics and §Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, February 25, 2003 , and in revised form, August 6, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In liver cells, the influx of Na+ mediated by nonselective cation (NSC) channels in the plasma membrane contributes importantly to regulation of cell volume. Under basal conditions, channels are closed; but both physiologic (e.g. insulin) and pathologic (e.g. oxidative stress) stimuli that are known to stimulate tyrosine kinases are associated with large increases in membrane Na+ permeability to ~80 pA/pF or more. Consequently, the purpose of these studies was to evaluate whether volume-sensitive tyrosine kinases mediate cell volume increases through effects on the activity or distribution of NSC channel proteins. In HTC hepatoma cells, decreases in cell volume evoked by hypertonic exposure increased total cellular tyrosine kinase activity ~20-fold. Moreover, hypertonic exposure (320–400 mosM) was followed after a delay by NSC channel activation and partial recovery of cell volume toward basal values (regulatory volume increase (RVI)). The tyrosine kinase inhibitors genistein and erbstatin prevented both NSC channel activation and RVI. Similarly, hypertonic exposure resulted in an increase in p60c-src activity, and intracellular dialysis with recombinant p60c-src led to activation of NSC currents in the absence of an osmolar gradient. Utilizing FM1-43 fluorescence, exposure to hypertonic media caused a rapid increase in the rate of exocytosis of ~40% (p < 0.01), and genistein inhibited both exocytosis and channel activation. These findings indicate that volume-sensitive increases in p60c-src and/or related tyrosine kinases play a key role in the regulation of membrane Na+ permeability, suggesting that increases in the NSC conductance may be mediated in part through rapid recruitment of a distinct pool of channel-containing vesicles.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver cell volume homeostasis is mandatory for cell survival. In addition, there is increasing evidence that cell volume represents a physiologically important mechanism for coupling changes in membrane transport to other cell and organ functions. Increases in cell volume, for example, serve as a signal to stimulate protein synthesis, bile secretion, and gene expression (1, 2). In addition, failure to regulate cell volume has been implicated in liver cell injury associated with alcohol, ischemia/reperfusion, and organ preservation (3, 4). Consequently, the definition of the sensing and regulatory mechanisms involved has broad implications for the modulation of liver cell and organ function as well as cellular response to injury (5).

Recent studies indicate that Na+-permeable ion channels in the plasma membrane play a key role in this process. Under basal conditions, membrane Na+ permeability is low because of tonic inhibition of channels by p38 mitogen-activated protein (MAP)1 kinase, a human homologue of the Saccharomyces cerevisiae HOG-1 gene product essential for cellular osmoregulation (6). Decreases in cell volume stimulated by oxidative stress and/or hypertonic exposure initiate an adaptive response that leads within minutes to Na+ influx through opening of nonselective cation (NSC) channels (5, 7). The resulting movement of water into the cell contributes to the restoration of volume toward basal values, a general process referred to as regulatory volume increase (RVI). Interestingly, hypertonic exposure has no effect on p38 MAP kinase activity, and the inhibitory effect of high intracellular concentrations of recombinant p38 MAP kinase can be overcome by changes in cell volume (6). Thus, it seems likely that inhibitory signaling is counterbalanced by additional, as yet unidentified pathways that increase the number or activity of NSC channels in the plasma membrane.

Both receptor and non-receptor tyrosine kinases play important roles in regulation of cell growth, cell cycle, and mitogenic signaling (8–10). In addition, there is increasing evidence that tyrosine kinases, either directly or through an effector pathway involving phosphoinositide (PI) 3-kinase, modulate vesicular exocytosis and related membrane transport events (10–13). These membrane effects include positive or negative modulation of Na+ influx through regulation of specific epithelial Na+ channels (ENaC) and transporters (NHE1) (14, 15). Interestingly, decreases in cell volume represent a potent stimulus for activation of tyrosine kinases in liver and other cell types (12, 16, 17), including autophosphorylation of insulin receptors and activation of members of the Src family of non-receptor tyrosine kinases (18, 19). Further, insulin receptor activation also leads to a robust exocytic response (11) and a primary increase in liver cell volume through opening of NSC channels with properties analogous to those responsible for RVI (2, 20). These findings suggest that, in liver, volume-sensitive tyrosine kinases may counter the inhibitory effects of p38 MAP kinase and lead to the opening of NSC channels and increases in cell volume. Based on these considerations, the purpose of these studies was to evaluate in a model liver cell line whether tyrosine kinases serve as positive signals that mediate RVI through effects on the activity and/or distribution of NSC channels.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Model—All studies were performed in HTC cells derived from rat hepatoma as described previously. These cells express insulin receptors, ion channels, and signaling pathways similar to those found in primary rat hepatocytes (5, 6, 21–23). Decreases in cell volume caused by exposure to hypertonic buffer or oxidative stress are followed by Na+ influx through opening of NSC channels in the plasma membrane (5, 6). Cells were passaged at weekly intervals and maintained at 37 °C, 5% CO2 in minimal essential media (Invitrogen) supplemented with heat-inactivated fetal bovine serum (10%) and L-glutamine (2 mM). Exposure of cells to hypertonic buffer (320–600 mosM, increase in NaCl) for 10 min had no effect on viability as measured by trypan blue exclusion and by intact and reversible whole-cell patch clamp current responses.

Measurement of Na+ Currents—Membrane Na+ currents were measured using whole-cell patch clamp techniques (24). Cells on a coverslip were mounted in a chamber (volume ~400 µl) and perfused at 4–5 ml/min with a standard extracellular solution containing (in mM): 140 NaCl, 4 KCl, 1 CaCl2, 2 MgCl2, 1 KH2PO4, 10 glucose, and 10 HEPES/NaOH (pH ~7.40). The standard intracellular (pipette) solution contained (in mM): 130 KCl, 10 NaCl, 2 MgCl2, 10 HEPES/KOH, 0.5 CaCl2, and 1 EGTA (pH 7.3), corresponding to a free [Ca2+] of ~100 nM (6). With these solutions, inward currents at a test potential of –80 mV are carried by influx of Na+ ions (5, 25). The ionic selectivity of currents was confirmed by partial substitution of extracellular Na+ with equimolar concentrations of K+ or Tris+ as indicated. Cell volume decreases were produced by exposure to hypertonic media created by addition to the extracellular media of (i) sucrose 20–50 mM or (ii) NaCl as indicated. Solution osmolality was measured by a vapor pressure osmometer (Westcor). Patch pipettes were pulled from Corning 7052 glass and had a resistance of 3–10 megaohms. Recordings were made with an Axopatch ID amplifier (Axon Instruments, Foster City, CA), and were digitized (1 kHz) for storage on a computer and analyzed using pCLAMP version 6.0 programs (Axon Instruments) as described (5, 6, 26). Current-voltage (I-V) relations were measured between –120 mV and +100 mV in 20 mV increments (400 ms duration, 2 s between test potentials); or using a ramp protocol (–100 mV to +100 mV in 400 ms). Pipette voltages (Vp) are referred to the bath. In the whole-cell configuration, Vp corresponds to the membrane potential, and downward deflections of the current trace indicate inward membrane current.

Reagents—Genistein and erbstatin at concentrations of 10 µM were utilized as nonselective inhibitors of cellular tyrosine kinases and were obtained from Calbiochem. PP2 at concentrations of 10 µM was utilized as a more selective inhibitor of p60c-src, and results were compared with the same concentrations of the inactive isomer PP3 (Calbiochem). Recombinant p60c-src was obtained from Calbiochem and was delivered to the cell interior by inclusion in the patch pipette solution and concentrations of 0.02 units/µl. {beta}-Mercaptoethanol (1%) and ATP (0.1 mM) were included in the same solution to increase specific activity as per the manufacturer's instructions. In control studies, intracellular dialysis with these co-factors in the absence of p60c-src did not activate currents. All other reagents were obtained from Sigma.

Measurement of Tyrosine Kinase Activities—Subconfluent cells in 100-mm tissue culture dishes were incubated at 37 °C for 10 min in medium alone or medium supplemented with 150 mM NaCl (600 mosM final). They were then washed three times in ice-cold isosmotic phosphate-buffered saline and lysed in 0.2–0.5 ml of ice-cold lysis buffer (50 mM {beta}-glycerophosphate (pH 7.2), 0.5% Triton X-100, 0.1 mM sodium vanadate, 2 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 2 µg/ml leupeptin, 4 µg/ml aprotinin). The lysate was centrifuged at 4 °C for 10 min (10,000 x g) to remove nuclei and cell debris, and the supernatants were adjusted to 100–200 µg of protein in 0.5 ml, to which was added 5 µl of rabbit polyclonal IgG against p60c-src (Santa Cruz Biotechnology, catalog no. sc-016, lot J135) and 100 µl of protein G-Sepharose (Amersham Biosciences). After 2 h of rocking incubation at 4 °C, the adsorbed proteins were washed three times in lysis buffer and resuspended in 40 µl of 50 mM {beta}-glycerophosphate (pH 7.2), 10 mM MgCl2, 0.1 mM sodium vanadate, 1 mM EGTA, and 0.1 mM [{gamma}-32P]ATP (5,000 cpm/pmol) containing 1 mM p60c-src substrate peptide (RRLIEDNEYTAR). The reactions were incubated for 20 min at 30 °C and then stopped by the addition of 10 µl of 25% trichloroacetic acid. The p60c-src substrate peptide phosphorylation was assessed by phosphocellulose filter binding as described previously (27). Total cellular tyrosine kinase activity was measured by Western blot using an anti-phosphotyrosine mouse IgG (Upstate Biotechnology Inc., catalog no. 05-321). Cultured cells were homogenized as described above, and lysates were heated in a boiling water bath after the addition of SDS sample buffer. Then 50 µg of protein was loaded per lane on a 10% polyacrylamide gel and subjected to SDS-PAGE. Proteins were transferred either to Immobilon or NitroPlus (MSI, Westboro, MA), and the blot was blocked with 5% BSA, Fraction V (Amresco, Solon, OH) in Tris-buffered saline, pH 8, plus 0.1% Tween 80 for 2 h at room temperature. Antiserum incubation was done at 4 °C for 16 h in 5% BSA/Tween 80/TBS, after which the membrane was washed with Tween 80/TBS. Secondary antisera conjugated to horseradish peroxidase was incubated with the membrane for 2 h at room temperature in 5% BSA/Tween 80/TBS followed by washes as described above and incubation with chemiluminescent substrate. The intensity of the bands on Western blot films was determined by scanning with a video image scanner and digitizing software.

For measurement of potential regulatory interactions between activated Akt and Src, cell cultures were exposed to hypertonic buffer (40% increase in NaCl, ~395 mosM) for 5 min in the presence of either the Src inhibitor PP2 (10 µM) or the PI 3-kinase inhibitor wortmannin (50 nM). Cell lysates were obtained after 0, 5, 15, and 30 min by the addition of 0.5 ml of lysis buffer (above). The lysates were centrifuged at 4 °C for 10 min at 21,000 x g to remove nuclei and cell debris. The supernatants were saved and used for Western blot analysis. Protein concentration was determined by the bicinchoninic acid method (Pierce). Immunoblotting was carried out using 10% polyacrylamide gels. Proteins were transferred by the procedures of Towbin and processed for ECL (Amersham Biosciences) for detection with specific antibodies using 5% BSA and 1% Tween in 1x TBS. All washes were in 0.5% Tween/TBS for 5 min. Analyses of autoradiograms were performed by video image scanning as described above. Anti-phospho-Src (Tyr-416) clone 9A6 monoclonal was obtained from Upstate USA, Inc., and anti-Phospho-Akt (Ser-473) was purchased from Cell Signaling Technology.

Cell Volume Measurements—Changes in cell volume were measured electronically using a Coulter Multisizer (Accucomp software, version 1.19, Hialeah, FL) with a 100 µM aperture and 20,000 cells/time point as described (6, 28). Changes in volume over time are expressed as relative volume normalized to the basal period in standard isotonic buffer.

Measurement of Membrane Turnover—The rate of exocytosis was assessed by real-time imaging using a fluorescent probe FM1-43 (Molecular Probes, OR) as described (11). FM1-43 has two specific properties that permit its use in this capacity. First, it binds to membranes but does not cross lipid bilayers. Second, it is not fluorescent in solution, but when it binds to biological membranes its quantum yield increases about 350-fold (29). Thus, the fluorescence intensity is directly proportional to the amount of membrane exposed to FM1-43. For these studies, FM1-43 was added to the external solution at a concentration of 4 µM. Initially, FM1-43 partitions into the plasma membrane exposed to the external solution (100%). Subsequently, when vesicles fuse with the plasma membrane, FM1-43 equilibrates with the new membrane resulting in an increase in the apparent fluorescence. Consequently, the overall change in FM1-43 fluorescence normalized to the initial plasma membrane value (100%) provides a measure of the sum of all exocytic events.

Statistics—Results are presented as the means ± S.E., with n representing the number of cells for patch clamp and imaging studies and the number of culture plates or repetitions for other assays. Same day controls were utilized for patch clamp and Coulter counter studies to minimize and effects of day-to-day changes in cell properties. Student's paired or unpaired t test was used to assess statistical significance as indicated, and p values < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Volume-dependent Increases in Tyrosine Kinase Activity—To evaluate whether tyrosine kinases are involved in the adaptive response to HTC cell shrinkage, the effects of hypertonic exposure on tyrosine kinase activity and cell volume were assessed, and the results are summarized in Fig. 1. Exposure of HTC cells to hypertonic buffer (150 mM increase in NaCl, ~600 mosM) for 10 min increased total tyrosine kinase activity ~20-fold, and the response was inhibited by genistein (10 µM, Fig. 1, top, n = 3, p < 0.01). In parallel studies, the effects of hypertonic exposure on cell volume were assessed by electronic cell sizing (20,000 cells/time point). Under control conditions, hypertonic exposure (40% increase in NaCl, ~395 mosM) caused a rapid initial decrease in relative cell volume (85.8 ± 2.1% of control value at 1 min) followed by a gradual recovery toward basal values (relative volume of 97.2 ± 2.2% at 25 min) despite the continued presence of hypertonic buffer (regulatory volume increase). In the presence of genistein (10 µM), there was a decrease in cell volume in isotonic buffer (relative volume of 93.2 ± 0.9%), an exaggerated response to hypertonic exposure (relative volume of 72.9 ± 2.2%), and no apparent RVI (relative volume of 76.1 ± 2.1% at 25 min; Fig. 1, bottom, p < 0.001 at all time points in hypertonic buffer). These findings confirm that tyrosine kinase activity is volume-sensitive in HTC cells as observed in primary hepatocytes (12) and suggest that it contributes to the regulation of cell volume recovery from shrinkage.



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  		FIG. 1.
Volume-sensitive tyrosine kinases are necessary for RVI. Top, hypertonic exposure increases total cellular tyrosine kinase activity. HTC cells were exposed to isotonic or hypertonic (~600 mosM, addition of NaCl) buffer solutions for 10 min and were then immediately homogenized in lysis buffer. Total tyrosine kinase activity was measured in triplicate as described under "Experimental Procedures." Genistein (10 µM) inhibits hypertonically induced tyrosine kinase activity (n = 3, p < 0.01). Bottom, inhibition of tyrosine kinase activity prevents cell volume recovery from shrinkage. HTC cell suspensions were exposed to hypertonic buffer (40% increase in NaCl, ~ 395 mosM) at time 0, and cell volume was measured with a Coulter Multisizer. Under control conditions (closed circles), hypertonic exposure resulted in a rapid initial decrease in cell volume followed by gradual recovery toward basal values. The addition of genistein (10 µM, open circles) decreased basal cell volume, exaggerated the response to hypertonic exposure, and inhibited RVI at all time points. Values represent means ± S.E. for 4 trials, with 20,000 cells for each time point.

 

Inhibition of Tyrosine Kinases Prevents Volume-dependent Activation of Na+ Influx—Previous studies indicate that RVI depends in large part on channel-mediated Na+ influx through a NSC conductance (5). The potential relationship between activation of tyrosine kinases and the increase in NSC conductance was evaluated using whole-cell patch clamp recording techniques; the results are summarized in Fig. 2. In the example shown in Fig. 2A, currents were measured at a holding potential of –40 mV and at test potentials of 0 mV and –80 mV (400 ms duration) at 10-s intervals; inward currents at –80 mV (downward deflection of the current trace) are carried by influx of Na+ ions through the NSC conductance pathway (6). Under basal conditions, Na+ currents (at –80 mV) were –13.2 + 3.3 pA (n = 16). Hypertonic exposure (20 mM sucrose, ~320 mosM) was followed after a delay by an increase in currents to –769.1 ± 63.5 pA at –80 mV (n = 16). To confirm that currents reflect Na+ influx through NSC channels, the current-voltage relationship was measured using a ramp protocol (Fig. 2B, –100 mV to +100 mV). With standard Na+-containing bath solutions, currents exhibited a nearly linear current-voltage relationship and reversed near the cation equilibrium potential (Ecat) of ~0 mV. Partial substitution of Na+ with K+ (final extracellular [K+] 140 mM, Ecat ~ –1.6 mV) had no significant effect on the reversal potential; but partial substitution of Na+ with the impermeable cation Tris+ (final extracellular [Na+]20 mM, Ecat –45.1 mV) decreased current density at –80 mV from –55.8 ± 5.9 to –4.45 ± 1.5 pA/pF and caused a negative shift in reversal potential to –40.4 ± 4.3 mV as anticipated for the NSC conductance (n = 5).



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  		FIG. 2.
Tyrosine kinase-dependent activation of an NSC conductance. A, representative whole-cell recording. Currents were measured at a holding potential of –40 mV and at test potentials of 0 mV and –80 mV at 10-s intervals. Currents at –80 mV (downward deflection of the current tracing) correspond to INa+ (see "Experimental Procedures"). Hypertonic exposure (sucrose 20 mM, ~ 320 mosM) resulted in activation of inward currents (upper tracing). In the presence of genistein (10 µM), the current response is inhibited (lower tracing). B, current-voltage relation of whole-cell currents. Utilizing a voltage ramp protocol (–100 mV to +100 mV over 400 ms) the currents induced by hypertonic exposure were measured. Under isotonic conditions currents were small (Na+, control). Hypertonic exposure resulted in large increases in current amplitude, characterized by reversal near 0 mV with either Na+ or K+ as the primary extracellular cation. When Na+ in the extracellular buffer solution was partially replaced with the Tris+ (final [Na+] = 20 mM), there was a significant decrease in inward current amplitude and a shift in reversal potential to –40.4 ± 4.3 mV as expected for a cation-selective channel. C, cumulative data recorded as average current density at –80 mV. Current density after hypertonic exposure (20 mM sucrose, ~320 mosM) was measured in the absence versus presence of the tyrosine kinase inhibitors (10 µM each) genistein (n = 6) and erbstatin (n = 6).

 

To evaluate whether tyrosine kinases contribute to activation of this conductance, cells were exposed to hypertonic buffer (~320 mosM) in the absence versus presence of putative tyrosine kinase inhibitors. Representative recordings are shown in Fig. 2A, and results are summarized in Fig. 2C. Under control conditions, hypertonic exposure increased current density to –86.3 ± 15.1 pA/pF (n = 10, p < 0.001). The peak response was markedly inhibited by prior exposure (preincubation for 15 min) to the tyrosine kinase inhibitors (10 µM each) genistein (–3.6 ± 0.4 pA/pF, n = 6, p < 0.01) or erbstatin (–27.6 ± 3.1 pA/pF, n = 6, p < 0.01). Thus, opening of the NSC channels may be mediated in part by activation of volume-sensitive tyrosine kinases.

p60c-src Represents a Candidate Volume-sensitive Tyrosine Kinase—p60c-src has been implicated in both positive and negative regulation of Na+ transport depending on the cell type and transport pathway under investigation (15). To evaluate whether p60c-src contributes to volume-sensitive Na+ influx in HTC cells, p60c-src activity was measured in cell lysates following high levels of hypertonic exposure to stimulate maximal activity as described under "Experimental Procedures." Exposure of cells to hypertonic buffer (600 mosM for 10 min) to stimulate maximal adaptive responses caused a 3-fold increase in specific p60c-src activity (Fig. 3A). Using more conventional levels of hypertonic exposure, preincubation of cells in PP2 (10 µM, 15 min), a cell-permeable inhibitor of p60c-src (30), inhibited current activation by hypertonic (40% increase in NaCl, ~407 mosM) exposure (Fig. 3B). Under control conditions, hypertonic exposure increased current density at –80 mV to –90.1 ± 11.9 pA/pF (n = 4). In the presence of PP2, the response was inhibited to –13.0 ± 2.6 pA/pF (n = 7, p < 0.001). Preincubation with the inactive analog PP3 (10 µM) had no effect (–93.5 ± 11.5 pA/pF, n = 4, NS). Thus, p60c-src represents one candidate for a tyrosine kinase involved in activation of Na+ influx through effects on the NSC conductance.



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  		FIG. 3.
p60c-src as a candidate tyrosine kinase involved in NSC activation. A, HTC cells were exposed to isotonic or hypertonic buffer (~600 mosM) for 10 min. Cells were then lysed and Src activity measured in lysates by immunoprecipitation assay as described under "Experimental Procedures." Data are reported as cpm of 32P incorporated into Src substrate peptide ("Experimental Procedures"; n = 3). B, cumulative data from whole-cell patch clamp recordings. Current density was measured at –80 mV. Under control conditions, hypertonic exposure increased current density (n = 4). Preincubation with the p60c-src inhibitor, PP2 (10 µM), inhibited current activation (n = 7, p < 0.001), but the inactive analog, PP3, had no effect (n = 4).

 

Intracellular Dialysis with p60c-src Activates NSC Channels in the Absence of a Volume Challenge—To assess whether p60c-src is capable of activating currents directly, whole-cell currents were measured in cells dialyzed with recombinant p60c-src. For these studies, p60c-src was included in the standard intracellular patch pipette solution (0.02 units/µl with 1% {beta}-mercaptoethanol and 0.1 mM ATP), and no osmotic gradients were imposed. Intracellular delivery of p60c-src resulted in the gradual activation of inward currents, which reached maximal values 2–5 min after achieving the whole-cell configuration (Fig. 4A). Partial substitution of extracellular Na+ with Tris+ again caused a decrease in inward currents (from –717.2 ± 60.1 to –69.1 ± 12.3 pA at –80 mV) and a negative shift in reversal potential (–41.2 ± 3.5 mV) toward the new cation equilibrium potential (Fig. 4B), indicating that inward p60c-src-activated currents are carried by influx of Na+ ions through the NSC conductance. The results summarized in Fig. 4C demonstrate that intracellular dialysis with p60c-src, but not heat-inactivated p60c-src, increases current density from –2.85 ± 1.1 to –35.7 ± 10.5 pA/pF (n = 8, p < 0.01). Thus, p60c-src is capable of stimulating Na+ influx in the absence of a primary volume decrease, overcoming the inhibitory effect of constitutive p38 MAP kinase activity.



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  		FIG. 4.
Intracellular dialysis with recombinant p60c-src leads to activation of NSC conductance in the absence of an osmolar gradient. A, representative whole-cell recording. The time of access to the cell interior is shown by the arrow. Currents were measured at a holding potential of –40 mV and at test potentials of 0 mV and –80 mV at 10-s intervals. Inclusion of recombinant p60c-src in the patch pipette solution resulted in the gradual activation of inward currents carried by Na+ (downward deflection of the current tracing). B, I-V relationship of whole-cell currents measured in response to intracellular dialysis with p60c-src. With Na+ as the primary extracellular cation, currents exhibited a nearly linear current-voltage relationship and reversal near 0 mV. Partial replacement of Na+ with Tris+ (final [Na+] = 20 mM) decreased inward currents and shifted the reversal potential to –41.2 ± 3.5 mV as expected for a cation-selective conductance. C, cumulative data recorded as average current density at –80mV. Intracellular dialysis with active p60c-src (0.02 units/µl; and the co-activators {beta}-mercaptoethanol and ATP as described under "Experimental Procedures") resulted in a large increase in current density, whereas inactive p60c-src (100 °C for 30 min) had no effect (n = 8).

 

Tyrosine Kinases Modulate Vesicular Trafficking—By analogy with other channel proteins, volume-sensitive tyrosine kinases can increase NSC activity by direct phosphorylation of channel proteins and/or by increasing the number of channel proteins in the plasma membrane (31, 32). Notably, volume-sensitive tyrosine kinases have been shown to modulate the trafficking of epidermal growth factor receptors in liver cells (12); both insulin receptor and p60c-src activities are closely associated with PI 3-kinase, which is known to regulate exo- and endocytosis (11, 33). To assess the possibility that membrane trafficking events may contribute to the NSC response, two complementary approaches were utilized. First, using patch clamp techniques, NSC currents were measured during hypertonic exposure in the absence versus presence of wortmannin (50 nM), a PI 3-kinase inhibitor known to inhibit exocytosis in HTC cells (11, 34) (Fig. 5). Wortmannin resulted in a substantial inhibition of current density from –76.6 ± 5.2 pA/pF (n = 4) to –17.0 ± 6.0 pA/pF (n = 6, p < 0.001). Second, the effects of hypertonic exposure on membrane turnover were assessed using FM1-43 fluorescence (Fig. 6). Under basal conditions in the presence of FM1-43, fluorescence intensity increases at a rate of 2.03 ± 0.07%/min as a result of constitutive exocytosis and continuous exposure of new vesicular membranes to FM1-43 in the bath (11). Exposure of cells to hypertonic media increased the rate of exocytosis to 2.84 ± 0.21% min–1 (n = 5, p < 0.01). The response was completely inhibited in the presence of genistein (1.93 ± 0.11% · min–1, n = 4, p < 0.02) but was unaffected by the microtubule inhibitor colchicine (3.05 ± 0.43% · min–1, n = 5, NS). Genistein had no effect on the rate of constitutive exocytosis (2.05 ± 0.04% · min–1 (n = 4, NS). These results suggest that volume-sensitive tyrosine kinases are involved in rapid recruitment of a pool of vesicles to the plasma membrane and that both tyrosine kinases and PI 3-kinase are necessary for the complete current response.



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  		FIG. 5.
Evidence for phosphoinositide 3-kinase in volume-sensitive signaling. A, I-V relationship of whole-cell currents measured during hypertonic exposure utilizing a voltage ramp protocol (from –100mV to +100 mV). The PI 3-kinase inhibitor, wortmannin (50 nM), significantly inhibited hypertonically induced currents. B, cumulative data recorded as average current density at –80mV in control (n = 4) versus wortmannin (n = 6). In the presence of wortmannin, hypertonically induced currents were significantly inhibited (p < 0.001).

 


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  		FIG. 6.
Volume-sensitive tyrosine kinases stimulate an increase in the rate of exocytosis. The rate of membrane turnover was assessed using FM1-43 as described under "Experimental Procedures." A, initially, during isotonic conditions, FM1-43 partitions into the plasma membrane (representative cell in panel 1). Hypertonic exposure (350 mosM) causes vesicles to fuse with the plasma membrane, and FM1-43 equilibrates with the new membrane resulting in an increase in the apparent fluorescence (same cell, panel 2). B, the rate of exocytosis, as measured by an increase in cellular fluorescence, increases during hypertonic exposure. The time points represented at points 1 and 2 correspond to the representative cells shown in A. C, fluorescence rate, expressed as % change in plasma membrane/min–1, under different experimental conditions. Genistein had no effect on the rate of constitutive exocytosis but completely inhibited the increase in exocytosis rate caused by hypertonic exposure (n = 4).

 

Activation of tyrosine kinases and PI 3-kinases is closely coordinated in many cell types (10, 35). This relationship can involve parallel increases in activity, tyrosine kinase-dependent regulation of PI 3-kinase, or vice versa (10, 35–37). Two approaches were utilized to evaluate the sequence of events following hypertonic exposure. First, in biochemical studies, cells were incubated with the PI 3-kinase inhibitor wortmannin (50 nM), and the effects on p60c-src activity were assessed; cells also were incubated with the Src inhibitor PP2 (10 µM), and the effects on PI 3-kinase activity were assessed. In duplicate studies, wortmannin had no effect on tyrosine phosphorylation of p60c-src as measured by densitometric scanning of Western blots using anti-Tyr-416 antibodies; and PP2 had no effect on Akt phosphorylation as measured by densitometric scanning of Western blots using anti-Ser-473 antibodies ("Experimental Procedures"). Second, in patch clamp studies, the effects of wortmannin on current activation by intracellular dialysis with p60c-src were assessed using the approach illustrated in Fig. 4. If p60c-src is upstream of PI 3-K, then wortmannin would be expected to completely inhibit the response. This was not observed. Using same day controls, intracellular dialysis with p60c-src increased current density to –60.0 ± 9.7 pA/pF in control cells (n = 4) as compared with –34.1 ± 4.3 pA/pF in the presence of wortmannin (n = 6). Thus, p60c-src is capable of partial activation of currents even in the presence of wortmannin. Taken together, these findings support important roles for both signaling pathways in the response to hypertonic exposure, suggesting that activation of p60c-src and PI 3-kinase occurs through parallel and not sequential mechanisms.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Nonselective cation channels play a critical role in the regulation of liver cell volume. The principal findings of the present studies are that tyrosine kinase activity is increased in response to hypertonic exposure and that tyrosine kinases in general and p60c-src in particular are necessary and sufficient for stimulation of increases in NSC channel activity. Moreover, these effects are closely coupled to rapid recruitment of a pool of vesicles, suggesting that the effects of volume-sensitive tyrosine kinases are mediated in part through modulation of the distribution of channels between the plasma membrane and vesicular addresses (12). Consequently, tyrosine kinases may serve as a critical intermediary signal modulating the dynamic interactions between cell volume and the NSC channel opening necessary for rapid modulation of cell volume in response to changing physiologic demands.

Previous studies indicate that the rate of Na+ influx across the hepatocyte plasma membrane is low under basal conditions and is balanced by an equivalent rate of Na+ efflux through Na+/K+ pump activity to maintain intracellular [Na+] near 8 mM (38). Decreases in cell volume are followed after a delay by an increase in Na+ influx mediated by (i) a Na+-permeable channel, (ii) Na+/H+ exchange, and (iii) Na+/K+/2Cl– cotransport (7). Among these, the channel-mediated pathway is quantitatively the most important, particularly at higher transmembrane osmolar gradients (5, 7). Constitutive p38 MAP kinase activity appears to play an essential role in the maintenance of basal cell volume through tonic inhibition of Na+-permeable channels (6). However, these inhibitory effects can be overcome by both hormonal and osmotic stimuli (5, 7, 21), indicating a role for counter-regulatory pathways that increase Na+ influx 40-fold or more above basal levels. The resulting increase in membrane cation permeability provides the driving force for water influx and increases in cell volume (5, 7).

Several observations support the concept that volume-sensitive Na+ influx is mediated by tyrosine kinase-dependent activation of NSC channels. First, maneuvers that stimulate Na+ influx (e.g. insulin, hypertonic stress) are associated with rapid activation of cellular tyrosine kinases. Second, these events appear to be causally related because inhibition of tyrosine kinase activity by genistein, erbstatin, or PP2 prevents both volume-sensitive channel opening and RVI. Finally, stimulation of tyrosine kinase activity with recombinant p60c-src is capable of activating the same conductance in the nominal absence of an osmolar stimulus. These findings suggest that volume-sensitive tyrosine kinases serve as critical intermediary signals that couple cell volume to changes in membrane Na+ permeability through effects on NSC channels.

These findings are of interest because insulin also stimulates an increase in liver cell volume through activation of NSC-like channels (2, 20), and cell volume per se may represent a signal regulating antiproteolytic and other insulin-like responses (39). Thus, although tyrosine kinases have established roles in the regulation of cell growth, cell cycle, and mitogenic signaling (8–10), they also appear capable of rapid modulation of membrane transport and membrane recycling.

Assuming that these findings are relevant to cells in vivo, several implications and uncertainties merit emphasis. First, small changes in liver cell volume have been shown to activate multiple signaling pathways in addition to tyrosine kinase (e.g. c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and h-sgk) (5, 6, 22, 40). For example, elevation of cytosolic [Ca2+] is required for activation of both whole-cell and single NSC channel currents (5, 22). Thus, it is likely that other signaling pathways complement the stimulatory effects of tyrosine kinases under defined conditions. Although JNK and ERK are likely to contribute to the characteristic effects of cell volume on metabolism and gene expression, they do not appear to be responsible for activation of Na+ influx and RVI (6). Despite the potential contributions of these additional pathways, the observations that cellular tyrosine kinases are volume-activated and that their inhibition completely prevents activation of the NSC conductance and RVI support an essential regulatory role.

Second, the site of action of tyrosine kinases is not defined fully. Indeed, multiple target proteins, including receptors for epidermal growth factor and CD95, are tyrosine-phosphorylated following hypertonic exposure (12). However, hypertonic exposure increases the rate of exocytosis by ~40%; and inhibition of exocytosis by blockade of either tyrosine kinase or PI 3-kinase pathways prevents channel activation. These findings are consistent with the known close functional interactions between tyrosine kinases and PI 3-kinases (10), suggesting that one site of action of tyrosine kinases could be to alter the distribution of channel proteins between intracellular vesicles and the plasma membrane and increase the number of channel proteins available for opening. It is also possible that tyrosine kinases modulate membrane Na+ permeability through direct regulatory phosphorylation of channel proteins (15). This possibility remains untested because the molecular basis of the NSC conductance has not been defined, a necessary step for the demonstration of tyrosine phosphorylation and/or localization to one or more vesicular pools. Na+-permeable channels encoded by ENaC are detectable in liver cells and may be involved in RVI (41). However, ENaC channels are inhibited by low concentrations of amiloride, exhibit high selectivity for Na+ over K+, and have a low unitary conductance, properties that are not consistent with the endogenous NSC conductance (6, 22).

This dual requirement for both tyrosine kinases and PI 3-kinases is also consistent with the observation that insulin receptor stimulation mobilizes vesicles sufficient in number to replace ~25% of the plasma membrane within minutes (11). However, the insulin response differs in that exocytosis is sensitive to colchicine and displays different kinetics, with a rapid burst of exocytosis followed after 2–3 min by a return to constitutive levels (11). Further, hypertonic activation of tyrosine kinase and PI 3-kinase appears to occur through parallel and not sequential mechanisms, and each is required for full activation of the current response. This complexity is underscored by the presence in liver of multiple PI 3-kinase isoforms and the demonstration that many of the effects of PI 3-kinase are tyrosine kinase-independent and may be unrelated to trafficking (42). Thus, it is attractive to speculate that activation of the NSC conductance represents a multistep process, with parallel signals coordinating both a redistribution of channel proteins and changes in channel open probability. This regulatory scheme permits both fidelity and selectivity in rapid modulation of membrane Na+ permeability.

Finally, although p60c-src represents a candidate for the volume-sensitive tyrosine kinase involved in RVI, other tyrosine kinases are likely to be involved as well. Specifically, high osmolar gradients increased total tyrosine kinase activity ~20-fold but increased p60c-src activity only ~3-fold. Nonetheless, the src inhibitor PP2 was capable of preventing current activation; and recombinant Src was capable of activating currents in the nominal absence of transmembrane osmolar gradients. Thus, it seems likely that Src proteins mediate the conductance response to hypertonic exposure under these experimental conditions. Additional volume-sensitive tyrosine kinases might also be capable of regulating the NSC conductance or could target other elements of the adaptive response to hypertonic exposure, including phosphorylation and translocation of epidermal growth factor and CD95 receptor proteins (12).

Collectively, these findings support a pivotal role for tyrosine kinases as mediators of regulatory volume increase in liver cells, suggesting that p60c-src is a candidate for one of the kinases specifically involved. Decreases in cell volume initiate an adaptive response that involves an increase in tyrosine kinase and p60c-src activities that, in turn, stimulate exocytosis and opening of a NSC conductance, Na+ influx, and restoration of cell volume toward basal values. Thus, cellular tyrosine kinases appear to serve as critical intermediary signals modulating the dynamic interactions between cell volume and cation channel opening necessary for rapid modulation of cell volume in response to changing physiologic demands.


   FOOTNOTES
 
* These studies were supported in part by grants from the American Liver Foundation (to G. K.) and the AGA/American Digestive Health Foundation (to A. P. F.) and by National Institutes of Health Grants DK 43278 (to J. G. F.), DK 46082 (to J. G. F.), and DK 61480 (to A. P. F.). In addition, the Waterman Foundation provided important support. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Rm. 6412, Campus Box B158, University of Colorado Health Sciences Center, Denver, CO 80262. E-mail: greg.fitz{at}uchsc.edu.

1 The abbreviations used are: MAP, mitogen-activated protein; PI, phosphoinositide; pF, picofarad; NSC, nonselective cation; RVI, regulatory volume increase; ENaC, epithelial Na+ channel; TBS, Tris-buffered saline; BSA, bovine serum albumin; NS, not significant. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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