Involvement of Integrins in Osmosensing and Signaling toward Autophagic Proteolysis in Rat Liver
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m210699200
ISSN1083-351X
AutoresStephan vom Dahl, Freimut Schliess, Regina Reissmann, Boris Görg, Oliver H. Weiergräber, Mariana Kocalkova, Frank Dombrowski, Dieter Häussinger,
Tópico(s)Calpain Protease Function and Regulation
ResumoInhibition of autophagic proteolysis by hypoosmotic or amino acid-induced hepatocyte swelling requires osmosignaling toward p38MAPK; however, the upstream osmosensing and signaling events are unknown. These were studied in the intact perfused rat liver with a preserved in situ environment of hepatocytes. It was found that hypoosmotic hepatocyte swelling led to an activation of Src (but not FAK), Erks, and p38MAPK, which was prevented by the integrin inhibitory hexapeptide GRGDSP, but not its inactive analogue GRGESP. Src inhibition by PP-2 prevented hypoosmotic MAP kinase activation, indicating that the integrin/Src system is located upstream in the osmosignaling toward p38MAPK and Erks. Inhibition of the integrin/Src system by the RGD motif-containing peptide or PP-2 also prevented the inhibition of proteolysis and the decrease in autophagic vacuole volume, which is otherwise observed in response to hypoosmotic or glutamine/glycine-induced hepatocyte swelling. These inhibitors, however, did not affect swelling-independent proteolysis inhibition by phenylalanine. In line with a role of p38MAPK in triggering the volume regulatory decrease (RVD), PP-2 and the RGD peptide blunted RVD in response to hypoosmotic cell swelling. The data identify integrins and Src as upstream events in the osmosignaling toward MAP kinases, proteolysis, and RVD. They further point to a role of integrins as osmo- and mechanosensors in the intact liver, which may provide a link between cell volume and cell function. Inhibition of autophagic proteolysis by hypoosmotic or amino acid-induced hepatocyte swelling requires osmosignaling toward p38MAPK; however, the upstream osmosensing and signaling events are unknown. These were studied in the intact perfused rat liver with a preserved in situ environment of hepatocytes. It was found that hypoosmotic hepatocyte swelling led to an activation of Src (but not FAK), Erks, and p38MAPK, which was prevented by the integrin inhibitory hexapeptide GRGDSP, but not its inactive analogue GRGESP. Src inhibition by PP-2 prevented hypoosmotic MAP kinase activation, indicating that the integrin/Src system is located upstream in the osmosignaling toward p38MAPK and Erks. Inhibition of the integrin/Src system by the RGD motif-containing peptide or PP-2 also prevented the inhibition of proteolysis and the decrease in autophagic vacuole volume, which is otherwise observed in response to hypoosmotic or glutamine/glycine-induced hepatocyte swelling. These inhibitors, however, did not affect swelling-independent proteolysis inhibition by phenylalanine. In line with a role of p38MAPK in triggering the volume regulatory decrease (RVD), PP-2 and the RGD peptide blunted RVD in response to hypoosmotic cell swelling. The data identify integrins and Src as upstream events in the osmosignaling toward MAP kinases, proteolysis, and RVD. They further point to a role of integrins as osmo- and mechanosensors in the intact liver, which may provide a link between cell volume and cell function. Changes in cell hydration within a narrow, physiological range markedly affect carbohydrate and protein metabolism, hepatic bile flow, gene expression, and cellular susceptibility to environmental stress (1Häussinger D. Biochem. J. 1996; 313: 697-710Crossref PubMed Scopus (501) Google Scholar, 2Häussinger D. Lang F. Trends Pharmacol. Sci. 1992; 13: 371-373Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 3Burg M.B. Am. J. Physiol. 1995; 268: F983-F996Crossref PubMed Google Scholar, 4Lang F. Busch G.L. Ritter M. Völkl H. Waldegger S. Gulbins E. Häussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar, 5McManus M. Churchwell K.B. Strange K. N. Engl. J. Med. 1995; 333: 1260-1266Crossref PubMed Scopus (402) Google Scholar, 6Schliess F. Häussinger D. Biol. Chem. Hoppe-Seyler. 2002; 383: 577-583Crossref PubMed Scopus (88) Google Scholar). The regulation of cell function by cell hydration requires structures, which sense hydration changes (osmosensing) and intracellular signaling pathways toward effector sites (osmosignaling). Progress has been made in identifying signal transduction pathways linking cell volume changes to alterations in cell function (7Häussinger D. Schliess F. Biochem. Biophys. Res. Commun. 1999; 255: 551-555Crossref PubMed Scopus (74) Google Scholar). For example, the MAPK, 1The abbreviations used are: MAPK, mitogen-activated protein kinase; AV, autophagic vacuole; DMEM, Dulbecco's modified Eagle's medium; Erk, extracellular signal-regulated kinase; FAK, focal adhesion kinase; MBP, myelin basic protein; PBS, phosphate-buffered saline; PI, phosphoinositide; RVD, regulatory volume decrease. p38MAPK, activation is critical for creating volume-regulatory ion fluxes in response to hypoosmotic swelling in perfused rat liver (8vom Dahl S. Schliess F. Graf D. Häussinger D. Cell. Physiol. Biochem. 2001; 11: 285-294Crossref PubMed Scopus (42) Google Scholar) and HTC liver cells (9Feranchak A.P. Berl T. Capasso J. Wojtaszek P.A. Han J. Fitz J.G. J. Clin. Invest. 2001; 108: 1495-1504Crossref PubMed Scopus (42) Google Scholar). Likewise, proteolysis inhibition by cell swelling strongly depends on activation of the p38MAPK in perfused rat liver (10Häussinger D. Schliess F. Dombrowski F. vom Dahl S. Gastroenterology. 1999; 116: 921-935Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Specific inhibition of the p38MAPK abolishes the antiproteolytic effects exerted by hypoosmolarity and glutamine, but is without effect on cell swelling under these conditions (10Häussinger D. Schliess F. Dombrowski F. vom Dahl S. Gastroenterology. 1999; 116: 921-935Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Another component involved in swelling-dependent proteolysis inhibition is the microtubular system. Colchicine blocks hypoosmotic proteolysis inhibition, but not p38MAPK activation upon hypoosmolarity (11vom Dahl S. Dombrowski F. Schliess F. Pfeifer U. Häussinger D. Biochem. J. 2001; 354: 31-36Crossref PubMed Scopus (57) Google Scholar). These data show that the microtubule-dependent element in hydration-dependent proteolysis signaling is obviously localized downstream of p38MAPK activation. In contrast to agonists that cause changes of cell hydration, the antiproteolytic action of non-swelling amino acids, e.g. phenylalanine, resides on the activation of other signaling events, such as activation of mammalian target of rapamycin (mTOR) and p70 ribosomal S6 protein kinase (p70S6K kinase) (12Blommaart E.F.C. Luiken J.J.F.P. Meijer A.M. Histochem. J. 1997; 29: 365-385Crossref PubMed Scopus (216) Google Scholar, 13Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 14Blommaart E.F. Krause U. Schellens J.P. Vreeling-Sindelarova H. Meijer A.J. Eur. J. Biochem. 1997; 243: 240-246Crossref PubMed Scopus (731) Google Scholar), which can clearly be differentiated from the swelling-related antiproteolytic signaling cascade (15van Sluijters D.A. Dubbelhuis P.F. Blommaart E.F. Meijer A.J. Biochem. J. 2000; 351: 545-550Crossref PubMed Scopus (123) Google Scholar). Whereas in bacteria, plants, and fungi two-component histidine kinases were identified to be involved in sensing of and subsequent adaptation to adverse osmotic conditions (16Loomis W.F. Shaulsky G. Wang N. J. Cell Sci. 1997; 110: 1141-1145Crossref PubMed Google Scholar), the mechanisms of osmosensing in mammalian cells are far from being understood. Integrins are candidates to be involved in mechanotransduction, i.e. the conversion of a mechanical stimulus into covalent modifications of signaling components. Integrins are heterodimers with each subunit having a single transmembrane domain. They establish cell adhesion to the extracellular matrix and bind inside the cell to cytoplasmic proteins, which in turn interact with different signal transduction components and the cytoskeleton (for reviews see Refs. 17Aplin A.E. Howe A. Alahari S.K. Juliano R.L. Pharmacol. Rev. 1998; 50: 197-263PubMed Google Scholar and 18Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6955) Google Scholar). In normal liver, the most important integrins are α1β1, α5β1, and α9β1 (19Carloni V. Mazzocca A. Pantaleo P. Cordella C. Laffi G. Gentilini P. Hepatology. 2001; 34: 42-49Crossref PubMed Scopus (75) Google Scholar, 20Hsu S.L. Cheng C. Shi Y.R. Cancer Lett. 2001; 167: 193-204Crossref PubMed Scopus (34) Google Scholar, 21Torimura T. Ueno T. Kin M. Harada R. Nakamura T. Kawaguchi T. Harada M. Kumashiro R. Watanabe H. Avraham R. Sata M. Hepatology. 2001; 34: 62-71Crossref PubMed Scopus (76) Google Scholar). The present study investigates the role of integrins and Src in hypoosmotic signaling toward proteolysis inhibition and cell volume regulation in the isolated perfused rat liver, which most authentically represents the three-dimensional hepatocyte anchoring to the extracellular matrix, preserved cell polarity, intact cytoskeleton, and structural/functional cell-cell interactions. Using the integrin antagonistic peptide GRGDSP and the Src kinase inhibitor PP-2, an integrin-dependent activation of Src kinases was localized upstream of swelling-induced p38MAPK signaling toward inhibition of autophagy. In contrast, the antiproteolytic effect of phenylalanine, which does not involve cell swelling and p38MAPK does not depend on GRGDSP-sensitive integrin action and Src activation. Consistent with inhibition of osmosignaling toward p38MAPK, GRGDSP and PP2 effectively antagonize the volume regulatory response triggered by hypoosmotic swelling. The findings suggest a role of integrins in hepatic osmosensing and transforming hepatocyte swelling into a physiological response. Liver Perfusion—Livers from male Wistar rats (160–230 g), fed a standard diet, were perfused in an open non-recirculating manner at a flow rate of 3.5–4.5 ml/min/g. The perfusion medium was bicarbonate-buffered Krebs-Henseleit saline plus lactate (2.1 mmol/liter) and pyruvate (0.3 mmol/liter), incubated with O2/CO2 (19:1, v/v), at a temperature of 37 °C. For changing the osmolarity, the NaCl concentration (115 mmol/liter) was varied, resulting in corresponding changes of osmolarity. Additions were made either by the use of micropumps (20 μl/min) or by dissolution in Krebs-Henseleit buffer. Inhibitors were dissolved in Me2SO. Viability of the livers was assessed by monitoring effluent oxygen concentration and measurement of lactate dehydrogenase leakage from livers, which did not exceed 15–20 milliunits/min/g of liver. Monitoring and Assays in Liver Perfusion—Effluent perfusate pH was monitored continuously with a pH-sensitive electrode, and the perfusion pressure was detected by a pressure transducer (Hugo Sachs Electronics, Hugstetten, Germany). Basal portal pressure was 3–5 cm H2O and was not affected by the compounds used in this study. The intracellular water space was calculated from the difference of washout profiles of simultaneously infused [14C]urea and [3H]inulin as described previously (22vom Dahl S. Hallbrucker C. Lang F. Gerok W. Häussinger D. Biol. Chem. Hoppe-Seyler. 1991; 372: 411-418Crossref PubMed Scopus (38) Google Scholar). In fed animals, the cell water under control conditions was 551 ± 10 μl/g (n = 28). Proteolysis was determined in separate perfusion experiments as 3H label release from rats that had been injected intraperitoneally with 50 μCi of l-4,5-[3H]leucine 16 h prior to the perfusion experiment as described previously (23Häussinger D. Hallbrucker C. vom Dahl S. Lang F. Gerok W. Biochem. J. 1990; 272: 239-242Crossref PubMed Scopus (83) Google Scholar). The rate of proteolysis was set to 100% under normotonic control conditions, due to different labeling of hepatic proteins after intraperitoneal injection, and the extent of inhibition of proteolysis was determined 30 min after institution of the respective condition, a time point, when a new steady state had been reached. In bile experiments, livers were perfused with 100 μmol/liter [3H]taurocholate (1 μCi/liter). Bile was collected at 2-min intervals. Bile flow was assessed by gravimetry, assuming a specific mass of 1 g/ml. Taurocholate excretion into bile was determined by liquid scintillation counting of the radioactivity present in bile, based on the specific radioactivity of [3H]taurocholate in influent perfusate. Preparation of Cultured Hepatocytes—Liver parenchymal cells were isolated from the livers of male Wistar rats (200 g body wt.) by collagenase perfusion as described previously by Meijer et al. (24Meijer A.J. Gimpel J.A. Deleeuw G.A. Tager J.M. Williamson J.R. J. Biol. Chem. 1975; 250: 7728-7738Abstract Full Text PDF PubMed Google Scholar). The cells were plated on fibronectin-coated culture dishes (17 μg/dish, diameter 60 mm, ∼1 × 106 cells) and maintained in Krebs-Henseleit buffer (KHB) with 6 mmol/liter glucose, equilibrated in a humidified atmosphere (air/CO2, 19:1, v/v) at 37 °C. After 4 h in KHB, the cells were cultured for another 48 h in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum and 1% penicillin/streptomycin, insulin (100 nmol/liter), 1% glutamine, dexamethasone (100 nmol/liter), sodium selenite (30 nmol/liter), and aprotinin (1 μg/ml). Fresh DMEM was added after 24 h. After a total cultivation time of 48 h, cells were cultured in normoosmotic DMEM without additions containing 1000 mg/liter glucose for 4 h. After starvation for 4 h in normoosmotic medium, cells were either exposed to hypoosmolar (205 mosmol/liter) or normoosmotic control medium (305 mosmol/liter) for 2 min. If indicated, cells were incubated with PP-2 (20 μmol/liter) or GRGDSP (250 μmol/liter) 20 min prior to installing hypoosmolarity or the normoosmotic control condition, respectively. At the end of experimental treatment, medium was removed from the culture, and cells were immediately lysed at 4 °C using lysis buffer containing 20 mmol/liter Tris-HCl (pH 7.4), 140 mmol/liter NaCl, 10 mmol/liter NaF, 10 mmol/liter sodium pyrophosphate, 1% Triton X-100, 1 mmol/liter EDTA, 1 mmol/liter EGTA, 1 mmol/liter sodium vanadate, 20 mmol/liter β-glycerophosphate, and protease inhibitor mixture (Roche Applied Science). The homogenized lysates were centrifuged at 20,000 × g at 4 °C, and protein analyses were performed as described below. Protein concentrations were determined according to Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Tissue Processing for Immune Complex Kinase Assays and Western Blot Analysis—Rat livers were perfused for 130 min with isoosmotic perfusion medium, thereafter with hypoosmotic perfusion medium (185 mosmol/liter). The desired osmolarity was achieved by omission of 60 mm NaCl. When indicated, inhibitors were present for 30 min prior to institution of hypoosmotic perfusion conditions or addition of amino acids. For immune complex assay and Western blot determinations, liver lobes from perfused liver were excised at the respective time points (0, 2, 5, 10, 20, and 30 min after installation of hypoosmotic perfusion conditions), dounced with an Ultraturrax (Janke & Kunkel, Staufen, Germany) at 0 °C in lysis buffer containing 20 mmol/liter Tris-HCl (pH 7.4), 140 mmol/liter NaCl, 10 mmol/liter NaF, 10 mmol/liter sodium pyrophosphate, 1% Triton X-100, 1 mmol/liter EDTA, 1 mmol/liter EGTA, 1 mmol/liter sodium vanadate, 20 mmol/liter β-glycerophosphate, and protease inhibitor mixture. Immune Complex Kinase Assays and Western Blot Analysis—The lysed samples from the perfused liver or hepatocytes were centrifuged at 4 °C, and aliquots of the supernatant were incubated with 1.5 μg of an antibody recognizing Erk-1 and Erk-2 for 2 h at 4 °C. Immune complexes were collected by using protein A-Sepharose 4B (Sigma), washed three times with lysis buffer and four times with kinase buffer (10 mmol/liter Tris-HCl (pH 7.4), 150 mmol/liter NaCl, 10 mmol/liter MgCl2, and 0.5 mmol/liter dithiothreitol), and incubated with 1 mg/ml MBP in the presence of 10 μCi [γ-32P]ATP for 30 min at 37 °C. The reactions were stopped by adding 2× gel loading buffer, and activity of Erk-2 was monitored via autoradiography after sodium dodecyl sulfatepolyacrylamide gel electrophoresis (12.5% gel). To perform SDS-PAGE and Western blot analysis an identical volume of 2× gel loading buffer containing 200 mmol/liter dithiothreitol (pH 6.8) was added to the lysates. After heating to 95 °C for 5 min, the proteins were subjected to SDS-PAGE (50 μg protein/lane, 7.5% gels). Following electrophoresis, gels were equilibrated with transfer buffer (39 mmol/liter glycine, 48 mmol/liter Tris-HCl, 0.03% SDS, 20% methanol). Proteins were transferred to nitrocellulose membranes using a semi-dry transfer apparatus (Amersham Biosciences). Blots were blocked overnight in 1% bovine serum albumin solubilized in 20 mmol/liter Tris-HCl, pH 7.5, containing 150 mmol/liter NaCl and 0.1% Tween 20 and then incubated for 3–4 h with antibodies raised against [Tyr(P)418]Src, [Tyr(P)529]Src, Src, [Tyr(P)397]FAK, FAK, [Thr(P)180/Tyr(P)182]p38MAPK and p38 at a dilution of 1:5,000. Following washing and incubation for 2 h with horseradish peroxidase-coupled anti-rabbit-IgG antibody (1:10,000), the blots were washed again and developed using enhanced chemiluminescent detection (Amersham GmbH, Freiburg, Germany). Densitometric analysis was performed with the E. A. S. Y. RH system (Herolab, Wiesloch, Germany). Electron Microscopy—For electron microscopic morphometry, fixation of the liver lobes was performed as described previously (11vom Dahl S. Dombrowski F. Schliess F. Pfeifer U. Häussinger D. Biochem. J. 2001; 354: 31-36Crossref PubMed Scopus (57) Google Scholar) by perfusion of glutaraldehyde (3%) in Krebs-Henseleit medium for 30 s. From the fixed livers small cubes of ∼1 mm3 were cut, postfixed for 2 h with 2% osmium tetroxide, 2% uranylacetate, and 1.5% lead citrate in PBS buffer, dehydrated in a graded series of ethanol and embedded in Epon 812. Thin sections for electron microscopy were placed on copper grids, stained with uranyl acetate and lead citrate, and were examined with a EM 900 electron microscope (Zeiss, Oberkochem, Germany). Quantitative Evaluation of Intracellular Organelles—The autophagic vacuoles were defined as bits of cytoplasm sequestered from the remaining cytoplasm by one or two membranes. The morphology of autophagic vacuoles has been described in detail elsewhere (26Pfeifer U. J. Cell Biol. 1978; 78: 152-167Crossref PubMed Scopus (237) Google Scholar). The square fields, which were defined by the copper grids (127 μm × 127 μm) were used as test fields and systematically searched for autophagic vacuoles at a magnification of ×10,500. The area of cytoplasm that was examined ranged between 7,000 and 12,000 μm2 (n = 6). The area of the AV was measured at magnification ×21,000. Low power electron micrographs of the test fields were mounted, and the area of the hepatocytic cytoplasm was calculated by count pointing method (144 test points). The fractional volume of autophagic vacuoles, which is defined as the volume of autophagic vacuoles per volume of liver cell cytoplasm (Vav/Vc) was calculated as described previously (10Häussinger D. Schliess F. Dombrowski F. vom Dahl S. Gastroenterology. 1999; 116: 921-935Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 27Pfeifer U. Lab. Invest. 1984; 50: 348-354PubMed Google Scholar). Immunocytochemistry and Confocal Laser Microscopy—For indirect immunofluorescence microscopy, rat livers were perfused for 120 min under isoosmotic conditions, and liver lobes were instantly fixed for cryosectioning in liquid nitrogen. When present, latrunculin B (2 μmol/liter) had been added 30 min prior to fixation of liver lobes. Liver sections were obtained using a cryotom CM 350 S (Leica, Bensheim, Germany) at a thickness of 5 μm. Air-dried samples were fixed using for 10 min at 4 °C and washed five times with ice-cold PBS. Immediately after washing samples were incubated with Phalloidin-FITC (Sigma) at a dilution of 1:500 in PBS containing 5% bovine serum albumine for 2 h at room temperature. Subsequently samples were washed again three times with ice-cold PBS. Immunostained liver perfusion samples were analyzed with a Leica TCS NT confocal laser scanning system (Leica, Bensheim, Germany) DM IRB inverted microscope. Images were acquired from a channel at a wavelength of 488 nm. Materials—The integrin antagonistic GRGDSP and the inactive control peptide GRGESP were from Bachem (Heidelberg, Germany). The antibody raised against Erk-1/Erk-2 was from Upstate (Charlottesville, VA). Antibodies recognizing [Tyr(P)397]FAK, [Tyr(P)418]Src, [Tyr(P)529]-Src and total Src were from BIOSOURCE (Camarillo, CA). Anti-[Thr(P)180/Tyr(P)182]p38MAPK antibody was from Promega (Madison, WI). The antibodies raised against total FAK and total p38 were from Santa Cruz Biotechnology. [γ-32P]ATP, l-[4,5-3H]leucine, [3H]inulin, and [14C]urea were from Amersham Biosciences. l-lactic acid was from Roth (Karlsruhe, Germany). Glutaraldehyde was purchased from Serva (Heidelberg, Germany). PP-2 was from Biomol (Plymouth, PA), and PP-3 and latrunculin B were from Calbiochem (Bad Soden, Germany). Dulbecco's modified Eagle's medium, fetal bovine serum, and gentamicin were purchased from Biochrom (Berlin, Germany). Fibronectin was purchased from Invitrogen (Karlsruhe, Germany). Enzymes were from Roche Applied Science. Insulin, dexamethasone, and glutamine were from Sigma. Penicillin/streptomycin was from Invitrogen. All other chemicals were from Merck (Darmstadt, Germany). Statistics—Data from different perfusion experiments are given as means ± S.E. (number of independent experiments). Conditions were compared by Student's t test. Differences were considered significant at p < 0.05. Involvement of Integrins and Src in Hypoosmotic Signal Transduction—Hypoosmotic (185 mosmol/liter) liver perfusion induced a transient Src phosphorylation on Tyr418, which was maximal (2.8 ± 0.6-fold, n = 4) at about 2 min. The increase in Src-Tyr418 phosphorylation was accompanied by a transient dephosphorylation of Src on Tyr529. According to earlier findings (10Häussinger D. Schliess F. Dombrowski F. vom Dahl S. Gastroenterology. 1999; 116: 921-935Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 28Schliess F. vom Dahl S. Häussinger D. Biol. Chem. Hoppe-Seyler. 2001; 382: 1063-1071Crossref PubMed Scopus (32) Google Scholar), hypoosmolarity induced a transient activation of the MAP kinases Erk-1/Erk-2 and p38, which was maximal between 5 and 10 min (Fig. 1). Normoosmotic control perfusions were without effect on Erk-1/Erk-2 and p38MAPK (10Häussinger D. Schliess F. Dombrowski F. vom Dahl S. Gastroenterology. 1999; 116: 921-935Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and neither altered Src phosphorylation on Tyr418 and Tyr529, nor FAK phosphorylation on Tyr397 (Fig. 1). Infusion of the integrin antagonistic peptide GRGDSP (10 μmol/liter), but not the inactive analogue GRGESP (10 μmol/liter) prevented the hypoosmotic stimulation of Src-Tyr418 phosphorylation and activation of the MAP kinases Erk-1/Erk-2 and p38 (Fig. 1B and Table 1). Likewise, PP-2 (250 nmol/liter), an inhibitor of Src kinases (29Hanke 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), abolished the hypoosmotic increase of Src-Tyr418 phosphorylation and activation of the MAP kinases (Fig. 1B and Table 1). In the presence of the inhibitors no significant effect of hypoosmotic perfusion on Src-Tyr529 phosphorylation could be observed (Table 1).Table IEffects of the integrin antagonistic peptide GRGDSP, its inactive analogue GRGESP and the Src inhibitor PP-2 on hypoosmotic Src-Tyr418 and dual phosphorylation of p38 and Erk-1/Erk-2 activation in perfused rat liverConditionInhibitor[Tyr(P)418]Src[Thr(P)180/Tyr(P)182]p38[Tyr(P)529]SrcErk-1/Erk-2HypoosmoticNone2.8 ± 0.6aResponse significantly different from isoosmotic control (p < 0.05)1.9 ± 0.2aResponse significantly different from isoosmotic control (p < 0.05)0.7 ± 0.1aResponse significantly different from isoosmotic control (p < 0.05)2.1 ± 0.2aResponse significantly different from isoosmotic control (p < 0.05)HypoosmoticGRGDSP1.2 ± 0.2bResponse significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p < 0.05)1.1 ± 0.1bResponse significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p < 0.05)0.7 ± 0.21.1 ± 0.1bResponse significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p < 0.05)HypoosmoticGRGESP2.1 ± 0.3aResponse significantly different from isoosmotic control (p < 0.05)2.7 ± 0.8cResponse different from the isoosmotic control (p = 0.06)0.6 ± 0.21.7 ± 0.1aResponse significantly different from isoosmotic control (p < 0.05)HypoosmoticPP-21.1 ± 0.2dResponse significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p = 0.07)1.1 ± 0.2bResponse significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p < 0.05)0.7 ± 0.21.1 ± 0.1bResponse significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p < 0.05)Data are from perfused rat liver exposed to hypoosmotic perfusion medium (185 mosmol/liter), without further pretreatment or in the presence of GRGDSP (10 μmol/liter), GRGESP (10 μmol/liter), or PP-2 (250 nmol/liter), as described in Fig. 1. Quantification was performed densitometrically. Phosphorylation of Src-Tyr418, Src-Tyr529, and p38-Thr180/Tyr182 and Erk-1/Erk-2 activity under isoosmotic conditions at the zero time point was set to one, and the maximal phosphorylation/activation observed following hypoosmotic exposure is given as a fraction thereof. Changes in Src-Tyr418 and Src-Tyr529 phosphorylation and dual p38 phosphorylation were normalized to expression of the respective total protein. Data are from 3-5 separate perfusion experiments and represent means ± S.Ea Response significantly different from isoosmotic control (p < 0.05)b Response significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p < 0.05)c Response different from the isoosmotic control (p = 0.06)d Response significantly different from the response evoked by hypoosmolarity in absence of inhibitory compounds (p = 0.07) Open table in a new tab Data are from perfused rat liver exposed to hypoosmotic perfusion medium (185 mosmol/liter), without further pretreatment or in the presence of GRGDSP (10 μmol/liter), GRGESP (10 μmol/liter), or PP-2 (250 nmol/liter), as described in Fig. 1. Quantification was performed densitometrically. Phosphorylation of Src-Tyr418, Src-Tyr529, and p38-Thr180/Tyr182 and Erk-1/Erk-2 activity under isoosmotic conditions at the zero time point was set to one, and the maximal phosphorylation/activation observed following hypoosmotic exposure is given as a fraction thereof. Changes in Src-Tyr418 and Src-Tyr529 phosphorylation and dual p38 phosphorylation were normalized to expression of the respective total protein. Data are from 3-5 separate perfusion experiments and represent means ± S.E Further experiments were performed in isolated liver cells plated on fibronectin. 48-h cultured cells were exposed to hypoosmotic (205 mosmol/liter) or normoosmotic (305 mosmol/liter) medium for 2 min. Hypoosmolarity induced an 2.7 ± 0.6-fold increase of Src-Tyr418 phosphorylation, which was blunted to 0.8 ± 0.1- and 1.1 ± 0.1-fold in the presence of GRGDSP (250 μmol/liter) or PP-2 (20 μmol/liter), respectively (n = 5). In a similar way, GRGDSP and PP-2 reduced the hypoosmotic p38 activation from 2.3 ± 0.4-fold under hypoosmotic control conditions to 0.9 ± 0.1- and 1.3 ± 0.2-fold (n = 5) in the presence of the respective inhibitors (Fig. 2). The findings support the suggestion that direct rather than indirect effects of GRGDSP and PP-2 account for the inhibitory effects found in the intact liver. The Integrin/Src System Is Involved in Regulatory Volume Decrease—As shown recently, p38MAPK activation in response to hypoosmotic cell swelling is also involved in regulatory volume decrease (RVD) (8vom Dahl S. Schliess F. Graf D. Häussinger D. Cell. Physiol. Biochem. 2001; 11: 285-294Crossref PubMed Scopus (42) Google Scholar), which manifests within about 10 min of hypoosmotic exposure as a net K+ and Cl- release from the hepatocytes through Ba2+-, DIDS-, and quinidine-sensitive ion channels (30Häussinger D. Stehle T. Lang F. Hepatology. 1990; 11: 243-254Crossref PubMed Scopus (63) Google Scholar). This RVD response only partially restores cell volume, and after its completion the cells are left in a slightly swollen state (30Häussinger D. Stehle T. Lang F. Hepatology. 1990; 11: 243-254Crossref PubMed Scopus (63) Google Scholar). When perfused livers are suddenly exposed to hypoosmotic fluid (225 mosmol/liter), a net K+ release of 12.2 + 0.5 μmol/g of liver is observed, which is completed within 415 ± 11 s (Table 3), and the residual cell volume increase after completion of RVD is 13.4 + 0.8% (Table 2). As shown recently (8vom Dahl S. Schliess F. Graf D. Häussinger D. Cell. Physiol. Biochem. 2001; 11: 285-294Crossref PubMed Scopus (42) Google Scholar), inhibition of p38MAPK blunts and delays this volume regulatory net K+ release and renders the cells in a more swollen state. As shown in Table 2, prevention of swelling-induced p38MAPK activation by GRGDSP or PP-2 rendered the cells in a significantly
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