Ca2+-dependent Protein Kinase C Isoforms Induce Cholestasis in Rat Liver
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m306242200
ISSN1083-351X
AutoresRalf Kubitz, Nirmalendu Saha, Thomas Kühlkamp, Supiya Dutta, Stephan vom Dahl, Matthias Wettstein, Dieter Häussinger,
Tópico(s)Trace Elements in Health
ResumoBile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca2+-dependent cPKCα and Ca2+-independent PKC∈ by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKC∈ fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors Gö6850 (1 μmol/liter) and Gö6976 (0.2 μmol/liter), a selective inhibitor of Ca2+-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca2+-dependent cPKCs, did not translocate rat PKC∈-YFP transfected in HepG2 cells. However, Ttx (0.5–10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. Gö6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca2+-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane. Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca2+-dependent cPKCα and Ca2+-independent PKC∈ by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKC∈ fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors Gö6850 (1 μmol/liter) and Gö6976 (0.2 μmol/liter), a selective inhibitor of Ca2+-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca2+-dependent cPKCs, did not translocate rat PKC∈-YFP transfected in HepG2 cells. However, Ttx (0.5–10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. Gö6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca2+-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane. Cholestasis is a major clinical issue and results from dys-regulation of transporter proteins in the sinusoidal (1Suzuki H. Sugiyama Y. Semin. Liver Dis. 2000; 20: 251-263Crossref PubMed Scopus (79) Google Scholar) and the canalicular membranes (2Keppler D. König J. Semin. Liver Dis. 2000; 20: 265-272Crossref PubMed Scopus (214) Google Scholar). Long term down-regulation of these transport systems involves changes in mRNA and protein levels (3Müller M. Semin. Liver Dis. 2000; 20: 323-337Crossref PubMed Scopus (51) Google Scholar, 4Trauner M. Meier P.J. Boyer J.L. N. Engl. J. Med. 1998; 339: 1217-1227Crossref PubMed Scopus (667) Google Scholar) while short term regulation is achieved by transporter retrieval and possibly covalent modification of transporter proteins (5Häussinger D. Schmitt M. Weiergräber O. Kubitz R. Semin. Liver Dis. 2000; 20: 307-321Crossref PubMed Google Scholar, 6Noé J. Hagenbuch B. Meier P.J. St Pierre M.V. Hepatology. 2001; 33: 1223-1231Crossref PubMed Scopus (102) Google Scholar). In some forms of cholestasis retrieval of transporter proteins precedes their expressional down-regulation (7Kubitz R. Wettstein M. Warskulat U. Häussinger D. Gastroenterology. 1999; 116: 401-410Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Although many inducers of short term cholestasis are known (7Kubitz R. Wettstein M. Warskulat U. Häussinger D. Gastroenterology. 1999; 116: 401-410Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 8Nathanson M.H. Gautam A. Bruck R. Isales C.M. Boyer J.L. Hepatology. 1992; 15: 107-116Crossref PubMed Scopus (68) Google Scholar, 9Kubitz R. D' Urso D. Keppler D. Häussinger D. Gastroenterology. 1997; 113: 1438-1442Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Schmitt M. Kubitz R. Wettstein M. vom Dahl S. Häussinger D. Biol. Chem. 2000; 381: 487-495Crossref PubMed Scopus (77) Google Scholar), the underlying signal transduction mechanisms are incompletely understood. Several studies related cholestasis to the action of protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; Bsep, bile salt export pump; Mrp2, multidrug resistance-associated protein 2; Ntcp, sodium taurocholate cotransporting polypeptide; cPKC, classical Ca2+-dependent protein kinase C; nPKC, novel Ca2+-independent protein kinase C; aPKC, atypical protein kinase C; MARCKS, myristoylated alanine-rich C kinase substrate; PMA, phorbol 12-myristate 13-acetate; Ttx, thymeleatoxin; ZO-1, zona occludens protein 1 associated with the tight junctions; YFP, yellow fluorescent protein; TC, taurocholate; TUDC, tauroursodeoxycholate. (11Corasanti J.G. Smith N.D. Gordon E.R. Boyer J.L. Hepatology. 1989; 10: 8-13Crossref PubMed Scopus (57) Google Scholar, 12Beuers U. Probst I. Soroka C. Boyer J.L. Kullak-Ublick G.A. Paumgartner G. Hepatology. 1999; 29: 477-482Crossref PubMed Scopus (69) Google Scholar, 13Kubitz R. Huth C. Schmitt M. Horbach A. Kullak-Ublick G.A. Häussinger D. Hepatology. 2001; 34: 340-350Crossref PubMed Scopus (77) Google Scholar). The first PKC isoform was isolated by Nishizuka (14Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5764) Google Scholar). Subsequently, 12 isoforms were characterized and grouped into classical, Ca2+-dependent (cPKC: α, βI, βII, γ), novel, Ca2+-independent (nPKC: ∈, δ, θ, η), and atypical (aPKC: ζ, ι) PKCs. In rat hepatocytes expression of PKCα, βII, δ, ∈ and ζ was demonstrated (15Perletti G.P. J. Biochem. Biophys. Methods. 1994; 28: 195-204Crossref PubMed Scopus (11) Google Scholar, 16Croquet F. Brehier A. Gil S. Davy J. Feger J. Biochim. Biophys. Acta. 1996; 1315: 163-168Crossref PubMed Scopus (21) Google Scholar). Stimulation of the Ca2+-independent PKC∈ isoform by taurolithocholate was reported to decrease bile flow (12Beuers U. Probst I. Soroka C. Boyer J.L. Kullak-Ublick G.A. Paumgartner G. Hepatology. 1999; 29: 477-482Crossref PubMed Scopus (69) Google Scholar), while the therapeutically used bile acid tauroursodeoxycholate (TUDC) increases bile flow (17Kurz A.K. Graf D. Schmitt M. vom Dahl S. Häussinger D. Gastroenterology. 2001; 121: 407-419Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 18Schliess F. Kurz A.K. vom Dahl S. Häussinger D. Gastroenterology. 1997; 113: 1306-1314Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The choleretic effect of TUDC is mediated by MAP kinases of the Erk-type (18Schliess F. Kurz A.K. vom Dahl S. Häussinger D. Gastroenterology. 1997; 113: 1306-1314Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and by the p38MAPK (17Kurz A.K. Graf D. Schmitt M. vom Dahl S. Häussinger D. Gastroenterology. 2001; 121: 407-419Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), while an anticholestatic effect of TUDC was attributed to the activation of the Ca2+-dependent PKCα (19Beuers U. Bilzer M. Chittattu A. Kullak-Ublick G.A. Keppler D. Paumgartner G. Dombrowski F. Hepatology. 2001; 33: 1206-1216Crossref PubMed Scopus (220) Google Scholar, 20Beuers U. Throckmorton D.C. Anderson M.S. Isales C.M. Thasler W. Kullak-Ublick G.A. Sauter G. Koebe H.G. Paumgartner G. Boyer J.L. Gastroenterology. 1996; 110: 1553-1563Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Thus, controversy exists regarding the role of individual PKC isoforms in bile secretion. In this study, the effects of PKC isoforms on bile formation were studied. Ca2+-dependent PKCs were found to trigger cholestasis and (in contrast to previous data (19Beuers U. Bilzer M. Chittattu A. Kullak-Ublick G.A. Keppler D. Paumgartner G. Dombrowski F. Hepatology. 2001; 33: 1206-1216Crossref PubMed Scopus (220) Google Scholar, 20Beuers U. Throckmorton D.C. Anderson M.S. Isales C.M. Thasler W. Kullak-Ublick G.A. Sauter G. Koebe H.G. Paumgartner G. Boyer J.L. Gastroenterology. 1996; 110: 1553-1563Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar)) were not involved in the choleretic response to TUDC. Materials—Phorbol 12-myristate 13-acetate (PMA), Gö6976, and thymeleatoxin were from Calbiochem (Bad Soden, Germany), Gö6850 and the mouse anti MRP2/Mrp2 (M2III-6) antibody were from Alexis (Grünberg, Germany). The rabbit anti-rat Bsep antibody (K12) and the rabbit anti-rat Ntcp (K4) were generous gifts from Dr. B. Stieger and Prof. P. Meier-Abt (Kantonsspital Zürich, Switzerland). The anti-mouse ZO-1 antibody was from Biozol (Eching, Germany). Isoform-specific PKC antibodies were from BD Biosciences (Heidelberg, Germany). The plasmid MARCKS-GFP was generously provided by Prof. N. Saito (Kobe University, Japan) (21Ohmori S. Sakai N. Shirai Y. Yamamoto H. Miyamoto E. Shimizu N. Saito N. J. Biol. Chem. 2000; 275: 26449-26457Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Rat Liver Perfusion—Livers of male Wistar rats (150 g) were perfused as described previously (18Schliess F. Kurz A.K. vom Dahl S. Häussinger D. Gastroenterology. 1997; 113: 1306-1314Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) in the presence of 100μmol/liter of taurocholate. Bile was collected every 2 min. Liver viability was assessed from the measurements of portal pressure, pH, and LDH- and glucose-release. After 30 min of perfusion, inhibitors or agonists were added to the medium as indicated. At various time points liver specimens were excised and snap frozen for cryosections or Western blots as described in Refs. 7Kubitz R. Wettstein M. Warskulat U. Häussinger D. Gastroenterology. 1999; 116: 401-410Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar and 9Kubitz R. D' Urso D. Keppler D. Häussinger D. Gastroenterology. 1997; 113: 1438-1442Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar. Isolation and Culture of Rat Hepatocytes—Rat hepatocytes were prepared as described previously (18Schliess F. Kurz A.K. vom Dahl S. Häussinger D. Gastroenterology. 1997; 113: 1306-1314Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Hepatocytes were cultured for 24 h in Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum, 6 mmol/liter glucose, 100 units/ml penicillin, 100 μg/ml streptomycin, 100 nmol/liter dexamethasone, and 100 nmol/liter insulin). FBS was omitted 12 h before experiments were started. Western Blot Analysis and Densitometry—Liver specimen were minced in homogenization buffer containing Tris (20 mmol/liter, pH 7.4), sucrose (250 mmol/liter), EGTA (5 mmol/liter), MgCl2 (1 mmol/liter), and protease inhibitors according to Ref. 12Beuers U. Probst I. Soroka C. Boyer J.L. Kullak-Ublick G.A. Paumgartner G. Hepatology. 1999; 29: 477-482Crossref PubMed Scopus (69) Google Scholar. Samples were ultracentrifuged for 60 min at 150,000 × g. The pellets were enriched in cell membrane-bound proteins while supernatants contained cytosolic proteins. Equal protein amounts of the membranous or cytosolic fractions were separated by SDS-PAGE. PKC isoforms were detected by specific monoclonal antibodies. Densitometry was performed with 1D Image Analysis Software (PerkinElmer Optoelectronics, Wiesbaden, Germany). The amount of membrane-bound (mb) PKC isoforms is expressed as the intensity ratio Imb/(Imb + Icytosolic) or as the x-fold change compared with control. The total amount of PKC in cultured rat hepatocytes was determined in whole cell lysates. Uptake Studies in Isolated Hepatocytes—Rat hepatocytes cultured for 12 h in fetal calf serum-free medium were treated with PMA, Ttx, or Me2SO (control) for 30 min and were then incubated for 2–10 min in fresh medium containing 150 μmol/liter taurocholate (TC) and 450 cpm/fmol [3H]TC. Uptake was stopped by removing the medium and washing the cells three times with ice-cold phosphate-buffered saline. Cells were lysed in a buffer containing 0.2 mol/liter NaOH and 0.05% SDS. Radioactivity of supernatants and cell lysates were measured in a scintillation counter (Packard instruments, Frankfurt, Germany). Unspecific binding of taurocholate was determined by incubating the cells in ice-cold incubation medium for 5 s. These values were subtracted from the uptake rates. Protein concentrations in cell lysates were determined with the Bradford method. TC uptake is expressed as pmol/mg protein. Uptake and unspecific binding was measured in three dishes per condition per cell preparation. Cloning of the Rat PKC∈ and Human PKCα and Transfection into HepG2 Cells—In order to clone the rat PKC∈ or the human PKCα, respectively, reverse transcription was performed using SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) and oligo(dT) as a primer. The templates were mRNA from rat brain and from HepG2 cells, respectively. For the subsequent PCR the forward primers were 5′-ATCCGCTAGCGAATTCCGGAATCCGGCGAG-3′ and 5′-ATCCGCTAGCGGAGCAAGAGGTGGTTGG-3′, respectively. They contained an NheI restriction site and started in front of the 5′-untranslated region of rat PKC∈ and human PKCα, respectively (according to the sequence with the accession numbers M18331 and NM002737.1, respectively). The reverse primers were 5′-CCGCGGTACCCAGGGCATCAGGTCTTCACC-3′ and 5′-CCGCGGTACCCATACTGCACTCTGTAAG-3′, respectively, where the stop codons were replaced by KpnI restriction sites. The resulting PCR products were cloned into the pGEM-T vector (Promega, Mannheim, Germany). After propagation in Escherichia coli inserts were excised with NheI and KpnI and were ligated into the vector pEYFP-N1 (Clontech, Palo Alto, CA) in frame to the N terminus of the enhanced yellow fluorescent protein. The sequences of the resulting plasmids (PKC∈-YFP and PKCα-YFP) were confirmed by sequencing. HepG2 cells were cultured in Dulbecco's modified Eagle's medium-Nutrimix F12 with 10% fetal calf serum. PKC∈-YFP, PKCα-YFP, or MARCKS-GFP were transfected into HepG2 cells with LipofectAMINE 2000 according to the manufacturer's guidelines (Invitrogen). 24–48 h later cells were analyzed by confocal microscopy. Confocal Laser Scanning Microscopy—Immunostaining and confocal laser-scanning microscopy were performed as described in Ref. 7Kubitz R. Wettstein M. Warskulat U. Häussinger D. Gastroenterology. 1999; 116: 401-410Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar using a Leica TCS-NT confocal laserscanning system mounted on an inverted microscope. Identical settings were used to compare liver sections from different experimental conditions. Cross-talk of fluorochromes was excluded by the use of the acousto optical tunable filter. The entire depth of a section was scanned in 0.5-μm steps. The resulting stacks of pictures were mounted as single projections. HepG2 cells transfected with PKC∈-YFP, PKCα-YFP, or MARCKS-GFP were cultured on 12-mm glass cover slips. 24–48 h after transfection coverslips were placed in a bath holder at 37 °C and covered with medium. Cells were selected according to the expression of PKC∈-YFP, PKCα-YFP, or MARCKS-GFP. After brief adjustments of the microscope (excitation at 488 nm, emission at >510 nm), confocal pictures of HepG2 cells were taken in regular intervals (10 s to 1 min). After 2 min, PMA (100 nmol/liter) or Ttx (100 nmol/liter) were added to the medium, and cells were scanned for up to 60 min. Gö6976 (1 μmol/liter) or Gö6850 (1 μmol/liter) were added 30 min before microscopy was started. In the case of PKC∈ PMA was applied to the medium at the end of Ttx treatment to finally prove translocation of PKC∈-YFP. Statistics—Data were reproduced from at least three perfusions or cell preparations. Values are given as means ± S.D. (Western blots, uptakes) or S.E. (perfusions). The two-sided Student's t test was used for statistical analysis, with a p < 0.05 considered to be statistically significant. Phorbolester-induced Cholestasis—PMA (10 nmol/liter) induced cholestasis in perfused rat livers and reduced bile secretion by 77% from 3.25 ± 0.22 to 0.75 ± 0.36 μl/g liver/min (n = 4) after 60 min in the presence of 100 μmol/liter taurocholate (Fig. 1). Taurocholate excretion was similarly reduced by 78% from 201 ± 16 to 45 ± 21 nmol/g liver/min (n = 4) (Fig. 1) indicating that taurocholate was the major driving force for bile secretion in the experimental setting used. After 60 min of LDH release was 144 ± 19 (n = 3) and 100 ± 13 milliunits/g liver/min (n = 4) in the absence and presence of PMA, respectively, suggesting that PMA perfusion does not cause cell damage to the perfused livers. Gö6850 (bisindolylmaleimide I, 1 μmol/liter), a potent inhibitor of most Ca2+-dependent and Ca2+-independent PKC isoforms (22Gekeler V. Boer R. Uberall F. Ise W. Schubert C. Utz I. Hofmann J. Sanders K.H. Schachtele C. Klemm K. Grunicke H. Br. J. Cancer. 1996; 74: 897-905Crossref PubMed Scopus (115) Google Scholar) diminished the effect of PMA on bile and taurocholate secretion by about 50% (Fig. 1). Gö6850 had no significant effect on these parameters by itself (not shown). Gö6976, a carbazole derivate, is a selective inhibitor of Ca2+-dependent PKC isoforms with an IC50 for cPKCα of 2.3 nmol/liter and for cPKCβI of 6.2 nmol/liter (23Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar). nPKCδ and nPKC∈ are not inhibited by Gö6976 even at micromolar concentrations (23Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar). Gö6976 (0.2 μmol/liter) inhibited the PMA-induced cholestasis by 60% after 60 min of PMA-infusion (Fig. 1), suggesting that the PMA-dependent cholestasis is largely transmitted by Ca2+-dependent PKC isoforms. Ca2+-independent PKC isoforms such as nPKCδ and nPKC∈, which are activated by PMA, apparently contribute to cholestasis only to a smaller extent. Activation of PKC isoforms is associated with their translocation from the cytosol to the plasma membrane (24Shoji M. Girard P.R. Mazzei G.J. Vogler W.R. Kuo J.F. Biochem. Biophys. Res. Commun. 1986; 135: 1144-1149Crossref PubMed Scopus (71) Google Scholar, 25Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar, 26Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (837) Google Scholar). In order to investigate the effect of PMA on subcellular PKC localization, rat livers were separated into a membrane (M) and cytosolic fraction (C) as described under "Experimental Procedures." In control livers cPKCα was distributed in favor of the cytosol in contrast to nPKC∈ (Fig. 2A). Liver samples were taken before or 20 min after Me2SO (control) or PMA perfusion. Both, cPKCα and nPKC∈ translocated to the membrane fraction in PMA-treated but not in control livers (Fig. 2B). The effect of PMA on both PKC isoforms was statistically significant. The amount of cPKCα in the membrane fraction increased 2.3 ± 0.4-fold (n = 3; p < 0.05) and was stronger compared with nPKC∈ translocation (1.6 ± 0.4-fold increase; n = 3; p < 0.05)). Calcium-dependent PKCs Mediate Cholestasis—Ttx, a diterpene-derivate of mezerein from the plant Thymelea hirsuta, is a selective activator of Ca2+-dependent PKC isoforms (27Ryves W.J. Evans A.T. Olivier A.R. Parker P.J. Evans F.J. FEBS Lett. 1991; 288: 5-9Crossref PubMed Scopus (176) Google Scholar, 28Llosas M.D. Batlle E. Coll O. Skoudy A. Fabre M. Garcia de Herreros A. Biochem. J. 1996; 315: 1049-1054Crossref PubMed Scopus (25) Google Scholar). In the perfused rat liver, Ttx at a concentration of 0.5 nmol/liter induced cholestasis (Fig. 3) and reduced bile flow and taurocholate excretion by ∼80%. The cholestatic effect was dose-dependent (Table I) resulting in an almost complete cholestasis at a Ttx concentration of 10 nmol/liter, while no signs of liver damage were detectable as assessed by LDH release (Table I). Ttx at 0.5 nmol/liter induced a minor increase in portal pressure (Fig. 3), which was more pronounced with increasing concentrations, therefore further experiments were performed with low Ttx concentrations (0.5 nmol/liter). Initial glucose output was 1.8 ± 0,2 nmol/g liver/min (n = 4) in controls and declined during perfusion. It was not significantly affected by Ttx (Fig. 3). The pH was not affected by Ttx treatment. At the beginning it was 7.44 ± 0.02 and 7.45 ± 0.01 in control and Ttx livers, respectively, and increased to 7.49 ± 0.03 and 7.51 ± 0.01, respectively, after 60 min.Table IDose-dependent effect of thymeleatoxin on bile flowTtxDecrease of bile flow nLDH releasenmol/liter%milliunits/g/min019 ± 2 (3)144 ± 190.161 ± 14 (3)119 ± 300.578 ± 7 (5)144 ± 161.081 ± 11 (3)167 ± 485.086 ± 8 (3)163 ± 1710.093 ± 4 (8)158 ± 11 Open table in a new tab Gö6976 (200 nmol/liter) abolished the cholestatic effect of Ttx (Fig. 3). This finding strongly suggests that Ttx-induced cholestasis is mediated by a Ca2+-dependent PKC isoform. Gö6976 itself had no significant effect (Fig. 3). In Ttx-treated livers (1 nmol/liter), cPKCα but not nPKC∈ translocated to the membrane fraction (Fig. 4A), in line with selective activation of cPKCs by Ttx. At 10-fold higher Ttx concentrations selectivity was still observed. The amount of membrane-bound cPKCα was 2.2 ± 0.3-fold increased in Ttx livers compared with control. The ratio of membrane-bound PKCα to total cPKCα significantly increased from 0.47 ± 0.03 (control, n = 3) to 0.65 ± 0.06 (Ttx, n = 3; p < 0.05) (Fig. 4B). In contrast, the ratio of membrane-bound to total nPKC∈ was not increased (Fig. 4B; control: 0.55 ± 0.06, n = 3; Ttx: 0.51 ± 0.05, n = 3) after 60 min of Ttx perfusion. At this time point no significant changes in the distribution of cPKCβ (control: 0.55 ± 0.06; Ttx: 0.47 ± 0.01) or of nPKCδ (control: 0.66 ± 0.02; Ttx: 0.72 ± 0.07) were detectable (not shown). In the membrane fraction of Ttx-treated livers a second band of higher electrophoretic mobility was detected by the cPKCα antibody (Fig. 4B). In order to provide further evidence for the specificity of Ttx compared with PMA, the Ca2+-independent PKC∈ from rat and the Ca2+-dependent human PKCα were cloned as described in the methods section and were fused to the N terminus of the yellow fluorescent protein (YFP). Human HepG2 cells were transfected with PKC∈-YFP or PKCα-YFP and were analyzed 24 h later. In many unstimulated cells PKC∈-YFP already localized to the cell membrane to some extent (e.g. Fig. 5A). However, PMA (100 nmol/liter) always induced a strong and rapid translocation of PKC∈-YFP to the cell membrane within 1–2 min (Fig. 5A and movie PMA and PKCe-YFP under Supplementary Materials). In contrast, no translocation to the plasma membrane was observed for PKC∈-YFP when cells were treated with Ttx (100 nmol/liter) for 15 min, while PMA still exerted the translocation (Fig. 5B, and movie Ttx plus PMA and PKCe-YFP under Supplementary Materials). Activation of the Ca2+-dependent human PKCα as indicated by its translocation to the cell membrane was observed for both PKC activators, PMA (Fig. 5C) and Ttx (Fig. 5D). It was noted that PKCα translocated faster in response to PMA compared with Ttx and that PKC∈ translocated faster than PKCα in response to PMA. Taken together, the data are in line with a selective activation of cPKCs but not nPKCs by Ttx. Differential Inhibition of PKC Isoforms by Gö6850 and Gö6976 —In order to investigate the efficacy of the two PKC inhibitors Gö6850 and Gö6976 in living cells, translocation of myristoylated alanine-rich C kinase substrates (MARCKS) was studied in transfected HepG2 cells. In unstimulated cells MARCKS is localized at the cell membrane because of its myristoylation at the N terminus and its basic residues in the effector domain (29George D.J. Blackshear P.J. J. Biol. Chem. 1992; 267: 24879-24885Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of multiple residues in the effector domain in response to PKC activation introduces negative charges into MARCKS and eventually leads to translocation of MARCKS into the cytosol (21Ohmori S. Sakai N. Shirai Y. Yamamoto H. Miyamoto E. Shimizu N. Saito N. J. Biol. Chem. 2000; 275: 26449-26457Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Therefore subcellular localization of MARCKS was used as an indicator of PKC activity. In transfected HepG2 cells, both, PMA (Fig. 6A) and Ttx (Fig. 6B) induced translocation of MARCKS to the cytosol, indicating PKC activation by these compounds. Gö6850 inhibited PMA-(Fig. 6C) and Ttx- (Fig. 6D) induced MARCKS-translocation to the cytosol, indicating that Gö6850 affects all PMA- and Ttx-sensitive PKC isoforms. In contrast, Gö6976 only inhibited MARCKS translocation induced by Ttx (Fig. 6F) but not by PMA (Fig. 6E), which is in line with its inhibitory effect on Ca2+-dependent but not Ca2+-independent PKCs. The differential effects of PKC activators and inhibitors used in this study are summarized in Fig. 6G. TUDC-induced Choleresis Is Independent of cPKCs—Tauroursodeoxycholate (TUDC, 20 μmol/l) increased taurocholate excretion from 220 ± 8 to 280 ± 9 nmol/g liver/min (Fig. 7) as described earlier (17Kurz A.K. Graf D. Schmitt M. vom Dahl S. Häussinger D. Gastroenterology. 2001; 121: 407-419Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 18Schliess F. Kurz A.K. vom Dahl S. Häussinger D. Gastroenterology. 1997; 113: 1306-1314Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). When Gö6976 (200 nmol/liter) was added 20 min before TUDC, it did not affect the TUDC-dependent increase in TC excretion (Fig. 7). This suggests that Ca2+-dependent PKC isoforms are not essential for TUDC signaling toward choleresis. TC Uptake Is Not Impaired by PKC Activation in Isolated Hepatocytes—Taurocholate is taken up by the basolateral sodium taurocholate cotransporting polypeptide (Ntcp) and by organic anion-transporting polypeptides (Oatp) (30Meier P.J. Stieger B. Annu. Rev. Physiol. 2002; 64: 635-661Crossref PubMed Scopus (478) Google Scholar). It is secreted by the canalicular bile salt export pump (Bsep). In order to differentiate whether PKC stimulation affects the transporter at the sinusoidal or the canalicular membrane, uptake of taurocholate was determined in isolated rat hepatocytes. 12 to 24 h after cell isolation, Ntcp was localized at the basolateral and canalicular proteins such as Mrp2 at the apical membrane (Fig. 8A), demonstrating polarized localization of the transporters. Cells cultured for 12 h were incubated 1 to 8 h with Ttx (100 nmol/liter) or PMA (100 nmol/liter). After treatment total amounts of cPKCα were determined in whole cell lysates by Western blots. Expressional down-regulation indirectly verified activation of cPKCα (Fig. 8B) by Ttx and PMA. The Km of Ntcp for TC transport is ∼25 μmol (31Hagenbuch B. Stieger B. Foguet M. Lubbert H. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10629-10633Crossref PubMed Scopus (455) Google Scholar, 32Kuhn W.F. Gewirtz D.A. Am. J. Physiol. 1988; 254: G732-G740PubMed Google Scholar). In order to saturate transport TC-uptake was measured in the presence of 150 μmol/liter of TC. TC uptake was almost linear during the first 10 min (Fig. 8D). When cells were preincubated for 30 min with 1, 10, or 100 nmol/liter of Ttx or PMA, no inhibition of the initial TC uptake was observed compared with controls (Fig. 8D). Likewise shorter preincubation (2 min) with Ttx or PMA (100 nmol/liter each) did not decrease initial TC uptake (not shown). These data suggest that cPKCs in cultured hepatocytes are responsive to PMA or Ttx treatment, but do not affect TC uptake across the sinusoidal membrane. Effects of PMA and Ttx on Mrp2 and Bsep Localization—It was demonstrated recently that cholestasis can result from retrieval of canalicular transporter proteins into subapical vesicles (7Kubitz R. Wettstein M. Warskulat U. Häussinger D. 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