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• W1995490018 abstract "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. 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. 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 (1Bruck R. Haddad P. Graf J. Boyer J.L. Am. J. Physiol. 1992; 262: G806-G812PubMed Google Scholar, 2Lang F. Busch G.L. Ritter M. Volkl H. Waldegger S. Gulbins E. Haussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar). In addition, failure to regulate cell volume has been implicated in liver cell injury associated with alcohol, ischemia/reperfusion, and organ preservation (3Wondergem R. Davis J. Alcohol. Clin. Exp. Res. 1994; 18: 1230-1236Crossref PubMed Scopus (35) Google Scholar, 4Carini R. Autelli R. Bellomo G. Albano E. Biochem. Biophys. Res. Commun. 1994; 202: 360-366Crossref PubMed Scopus (24) Google Scholar). 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 (5Schlenker T. Feranchak A.P. Schwake L. Stremmel W. Fitz J.G. Gastroenterology. 2000; 118: 395-403Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). 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) 1The 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. kinase, a human homologue of the Saccharomyces cerevisiae HOG-1 gene product essential for cellular osmoregulation (6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar). 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 (5Schlenker T. Feranchak A.P. Schwake L. Stremmel W. Fitz J.G. Gastroenterology. 2000; 118: 395-403Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 7Wehner F. Tinel H. J. Physiol. 1998; 506: 127-142Crossref PubMed Scopus (61) Google Scholar). 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 (6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar). 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 (8Broome M.A. Hunter T. J. Biol. Chem. 1996; 271: 16798-16806Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Hunter T. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1998; 29: 583-605Crossref Scopus (364) Google Scholar, 10Blume-Jensen P. Hunter T. Nature. 2003; 411: 355-365Crossref Scopus (3161) Google Scholar). 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 (10Blume-Jensen P. Hunter T. Nature. 2003; 411: 355-365Crossref Scopus (3161) Google Scholar, 11Kilic G. Doctor R.B. Fitz J.G. J. Biol. Chem. 2001; 276: 26762-26768Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Reinher R. Schliess F. Haussinger D. FASEB J. 2003; 17: 731-733Crossref PubMed Scopus (100) Google Scholar, 13Tsunoda Y. Africa L. Steil G.J. Owyang C. Biochem. Biophys. Res. Commun. 1996; 23: 876-884Crossref Scopus (44) Google Scholar). These membrane effects include positive or negative modulation of Na+ influx through regulation of specific epithelial Na+ channels (ENaC) and transporters (NHE1) (14Krump E. Nikitas K. Grinstein S. J. Biol. Chem. 1997; 272: 17303-117311Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 15Gilmore E.S. Stutts M.J. Milgram S.L. J. Biol. Chem. 2001; 276: 42610-42617Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Interestingly, decreases in cell volume represent a potent stimulus for activation of tyrosine kinases in liver and other cell types (12Reinher R. Schliess F. Haussinger D. FASEB J. 2003; 17: 731-733Crossref PubMed Scopus (100) Google Scholar, 16Szaszi K. Buday L. Kapus A. J. Biol. Chem. 1997; 272: 16670-16678Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Rizoli S.B. Rotstein O.D. Kapus A. J. Biol. Chem. 1999; 274: 2072-22080Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), including autophosphorylation of insulin receptors and activation of members of the Src family of non-receptor tyrosine kinases (18Ouwens D.M. Gomes de Mesquita D.S. Dekker J. Maassen J.A. Biochim. Biophys. Acta. 2001; 1540: 97-106Crossref PubMed Scopus (28) Google Scholar, 19Kapus A. Szaszi K. Sun J. Rizoli S.B. Rotstein O.D. J. Biol. Chem. 1999; 274: 8093-8102Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Further, insulin receptor activation also leads to a robust exocytic response (11Kilic G. Doctor R.B. Fitz J.G. J. Biol. Chem. 2001; 276: 26762-26768Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and a primary increase in liver cell volume through opening of NSC channels with properties analogous to those responsible for RVI (2Lang F. Busch G.L. Ritter M. Volkl H. Waldegger S. Gulbins E. Haussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar, 20Al-Habori M. Peak M. Thomas T.H. Agius L. Biochem. J. 1992; 282: 789-796Crossref PubMed Scopus (45) Google Scholar). 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. 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 (5Schlenker T. Feranchak A.P. Schwake L. Stremmel W. Fitz J.G. Gastroenterology. 2000; 118: 395-403Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar, 21Lidofsky S.D. Xie M.H. Sostman A. Scharschmidt B.F. Fitz J.G. J. Biol. Chem. 1993; 268: 14632-14636Abstract Full Text PDF PubMed Google Scholar, 22Lidofsky S.D. Sostman A. Fitz J.G. J. Membr. Biol. 1997; 157: 231-236Crossref PubMed Scopus (20) Google Scholar, 23Fitz J.G. Sostman A. Middleton J.P. Am. J. Physiol. 1994; 266: G677-G684PubMed Google Scholar). 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 (5Schlenker T. Feranchak A.P. Schwake L. Stremmel W. Fitz J.G. Gastroenterology. 2000; 118: 395-403Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar). Cells were passaged at weekly intervals and maintained at 37 °C, 5% CO2 in HCO3--containing 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 (24Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15175) Google Scholar). 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 (6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar). With these solutions, inward currents at a test potential of –80 mV are carried by influx of Na+ ions (5Schlenker T. Feranchak A.P. Schwake L. Stremmel W. Fitz J.G. Gastroenterology. 2000; 118: 395-403Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 25Roman R.M. Wang Y. Lidofsky S.D. Feranchak A.P. Lomri N. Scharschmidt B.F. Fitz J.G. J. Biol. Chem. 1997; 272: 21970-21976Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). 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 (5Schlenker T. Feranchak A.P. Schwake L. Stremmel W. Fitz J.G. Gastroenterology. 2000; 118: 395-403Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar, 26Fitz J.G. Sostman A. Am. J. Physiol. 1994; 266: G544-G553PubMed Google Scholar). 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. β-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 β-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 × 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 β-glycerophosphate (pH 7.2), 10 mm MgCl2, 0.1 mm sodium vanadate, 1 mm EGTA, and 0.1 mm [γ-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 (27Heasley L.E. Senkfor S.I. Winitz S. Strasheim A. Teitelbaum I. Berl T. Am. J. Physiol. 1994; 267: F366-F373PubMed Google Scholar). 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 × 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 1× 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 (6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar, 28Roman R.M. Feranchak A.P. Davison A.K. Schwiebert E.M. Fitz J.G. Am. J. Physiol. 1999; 277: G1222-G1230Crossref PubMed Google Scholar). 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 (11Kilic G. Doctor R.B. Fitz J.G. J. Biol. Chem. 2001; 276: 26762-26768Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 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 (29Betz W.J. Mao F. Bewick G.S. J. Neurosci. 1992; 12: 363-375Crossref PubMed Google Scholar). 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. 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 (12Reinher R. Schliess F. Haussinger D. FASEB J. 2003; 17: 731-733Crossref PubMed Scopus (100) Google Scholar) and suggest that it contributes to the regulation of cell volume recovery from shrinkage. 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 (5Schlenker T. Feranchak A.P. Schwake L. Stremmel W. Fitz J.G. Gastroenterology. 2000; 118: 395-403Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). 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 (6Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Investig. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar). 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 (E cat) of ∼0 mV. Partial substitution of Na+ with K+ (final extracellular [K+] 140 mm, E cat ∼ –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, E cat –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). 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 (15Gilmore E.S. Stutts M.J. Milgram S.L. J. Biol. Chem. 2001; 276: 42610-42617Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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 (30Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar), 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. Intracellular Dialysis with p60c-src Activates NSC Channels in the Absence of a Volume Challenge—To assess whether p60 c-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% β-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. 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 (31Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 26" @default.
• W1995490018 created "2016-06-24" @default.
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• W1995490018 creator A5037262248 @default.
• W1995490018 creator A5042225850 @default.
• W1995490018 creator A5042663920 @default.
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• W1995490018 date "2003-11-01" @default.
• W1995490018 modified "2023-09-30" @default.
• W1995490018 title "Volume-sensitive Tyrosine Kinases Regulate Liver Cell Volume through Effects on Vesicular Trafficking and Membrane Na+ Permeability" @default.
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