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W1998248849 abstract "Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2- and 5-fold increase in K+ and Cl− currents, respectively, in HTC cells. These increases occurred within 5–10 min after TNF exposure and were inhibited either by K+ or Cl−substitution or by K+ channel blockers (Ba2+, quinine, 0.1 mm each) or Cl− channel blockers (10 μm 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mm N-phenylanthranilic acid), respectively. TNF-mediated increases in K+ and Cl− currents were each inhibited by intracellular Ca2+ chelation (5 mm EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 μm) or PKC 19–36 peptide (1 μm). In contrast, currents were not attenuated by the calmodulin kinase II 281–309 peptide (10 μm), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 μm), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K+ and Cl− channels is an early response to TNF signaling and that channel opening is Ca2+- and PKC-dependent. Our findings further suggest that K+ and Cl− channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF. Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2- and 5-fold increase in K+ and Cl− currents, respectively, in HTC cells. These increases occurred within 5–10 min after TNF exposure and were inhibited either by K+ or Cl−substitution or by K+ channel blockers (Ba2+, quinine, 0.1 mm each) or Cl− channel blockers (10 μm 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mm N-phenylanthranilic acid), respectively. TNF-mediated increases in K+ and Cl− currents were each inhibited by intracellular Ca2+ chelation (5 mm EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 μm) or PKC 19–36 peptide (1 μm). In contrast, currents were not attenuated by the calmodulin kinase II 281–309 peptide (10 μm), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 μm), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K+ and Cl− channels is an early response to TNF signaling and that channel opening is Ca2+- and PKC-dependent. Our findings further suggest that K+ and Cl− channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF. tumor necrosis factor calmodulin kinase II protein kinase C 5-nitro-2-(3-phenylpropylamino)benzoic acid N-phenylanthranilic acid cytosolic calcium concentration 4′,6-diaminido-2-phenylindole current at −80 mV current at 0 mV picofarad Tumor necrosis factor α (TNF)1 is an inflammatory cytokine that induces programmed cell death in a variety of tissue types (1.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar). In the liver, TNF has been implicated as a mediator of hepatocellular dysfunction and death following toxic injury, viral hepatitis, and sepsis (2.Czaja M.J. Xu J. Alt E. Gastroenterology. 1995; 108: 1849-1854Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 3.Iimuro Y. Gallucci R.M. Luster M.I. Kono H. Thurman R.G. Hepatology. 1997; 26: 1530-1537Crossref PubMed Scopus (446) Google Scholar, 4.Bird G.L. Sheron N. Goka A.K. Alexander G.J. Williams R.S. Ann. Intern. Med. 1990; 112: 917-920Crossref PubMed Scopus (430) Google Scholar, 5.Mizuhara H. O'Neill E. Seki N. Ogawa T. Kusunoki C. Otsuka K. Satoh S. Niwa M. Senoh H. Fujiwara H. J. Exp. Med. 1994; 179: 1529-1537Crossref PubMed Scopus (493) Google Scholar, 6.Ando K. Moriyama T. Guidotti L.G. Wirth S. Schreiber R.D. Schlicht H.J. Huang S.N. Chisari F.V. J. Exp. Med. 1993; 178: 1541-1554Crossref PubMed Scopus (414) Google Scholar, 7.Ando K. Hiroishi K. Kaneko T. Moriyama T. Muto Y. Kayagaki N. Yagita H. Okumura K. Imawari M. J. Immunol. 1997; 158: 5283-5291PubMed Google Scholar, 8.Jaeschke H. Fisher M.A. Lawson J.A Simmons C.A. Farhood A. Jones D.A. J. Immunol. 1998; 160: 3480-3486PubMed Google Scholar). It is thought that such pathological conditions lead to the release of TNF by hepatic macrophages, with resultant paracrine actions on other liver cells (9.Bradham C.A. Plumpe J. Manns M.P. Brenner D.A. Trautwein C. Am. J. Physiol. 1998; 275: G387-G392PubMed Google Scholar). In liver, TNF exhibits pleiotropic effects, ranging from reduction of bile flow to hepatocellular apoptosis (10.Whiting J.F. Green R.M. Rosenbluth A.B Gollan J.L. Hepatology. 1995; 22: 1273-1278PubMed Google Scholar, 11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 12.Kim Y.M. de Vera M.E Watkins S.C Billiar T.R. J. Biol. Chem. 1997; 272: 1402-1411Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). Experimental evidence supports several mechanisms to account for such effects, including activation of caspases and kinases, generation of free radicals, and down-regulation of membrane organic solute transporters (13.Gross A. Yin X.M. Wang K. Wei M.C. Jockel J. Milliman C. Erdjument-Bromage H. Tempst P. Korsmeyer S.J. J. Biol. Chem. 1999; 274: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar, 14.Ventura J.J. Roncero C. Fabregat I. Benito M. Hepatology. 1999; 29: 849-857Crossref PubMed Scopus (20) Google Scholar, 15.Fernandez-Checa J.C. Kaplowitz N. Garcia-Ruiz C. Colell A. Miranda M. Mari M. Ardite E. Morales A. Am. J. Physiol. 1997; 273: G7-G17Crossref PubMed Google Scholar, 16.Moseley R.H. Wang W. Takeda H. Lown K. Shick L. Ananthanarayanan M. Suchy F.J. Am. J. Physiol. 1996; 271: G137-G146PubMed Google Scholar, 17.Green R.M. Beier D. Gollan J.L. Gastroenterology. 1996; 111: 193-198Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Despite this body of evidence, there remain significant gaps in our knowledge regarding the responsible pathways that couple TNF to liver damage.There is abundant information to suggest that apoptosis is associated with increases in mitochondrial membrane permeability but considerably less is known with respect to the role of the plasma membrane. In a limited number of cell types, increases in plasma membrane permeability to ions represent early responses to apoptotic stimuli. In cultured neurons, serum withdrawal increases voltage-activated K+channel activity and cell death, and in lymphocytes, engagement of the cell surface protein Fas leads to K+ loss, activation of outwardly rectifying Cl− channels, and apoptosis (18.Yu S.P. Yeh C.H. Sensi S.L. Gwag B.J. Canzoniero L.M. Farhangrazi Z.S. Ying H.S. Tian M. Dugan L.L. Choi D.W. Science. 1997; 278: 114-117Crossref PubMed Scopus (532) Google Scholar, 19.Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 20.Szabo I. Lepple-Wienhues A. Kaba K.N. Zoratti M. Gulbins E. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6169-6174Crossref PubMed Scopus (197) Google Scholar). Conversely, pharmacological blockade of K+ or Cl− channels blunts apoptosis in these experimental models. Thus, K+ and Cl− channels may play important roles in apoptotic processes.Based on its function in inducing apoptosis, there is reason to believe that TNF could influence cell death through activation of ion channels. In support of this, TNF increases K+ currents in selected neurons and activates Cl− currents in neutrophils (21.Houzen H. Kikuchi S. Kanno M. Shinpo K. Tashiro K. J Neurosci. Res. 1997; 50: 990-999Crossref PubMed Scopus (66) Google Scholar,22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar). However, it is unknown whether TNF affects plasma membrane ion channels in other cell types or whether such channels are involved in TNF-mediated cell death. In this study, we have examined the effects of TNF on plasma membrane conductance and death in the model hepatocyte-like cell line HTC. Our results suggest that TNF activates K+ and Cl− channels and that channel activation is an early signal in pathways leading to TNF-mediated liver cell death.DISCUSSIONIn this study, we have examined the role of plasma membrane ion channels in TNF signaling. Our observations suggest that TNF increases membrane permeability to K+ and Cl− in HTC cells through activation of ion channels and that TNF-mediated cell death is delayed by blockade of these channels. Our data are thus consistent with the hypothesis that channel opening is an early event in a TNF-mediated pathway that leads to liver cell death.Two principal findings support the concept that TNF activates K+ and Cl− channels. First, TNF increased membrane currents (I0 and I−80) at potentials corresponding to the opening of K+ and Cl−channels. Second, the TNF-evoked increases in these currents were selectively prevented by either K+ or Cl−removal or by exposure to K+ or Cl− channel blockers, respectively. Our data thus extend observations in neurons, in which TNF activates K+ channels (21.Houzen H. Kikuchi S. Kanno M. Shinpo K. Tashiro K. J Neurosci. Res. 1997; 50: 990-999Crossref PubMed Scopus (66) Google Scholar), and neutrophils, in which TNF activates Cl− channels (22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar), and demonstrate that TNF can activate K+ and Cl− channels in the same cell type.Unexpectedly, the TNF-elicited increase in K+ current (I0) was inhibited by Cl− removal. However, this current was not attenuated by Cl− channel blockers. This suggests that K+ channel opening does not depend on Cl− movement per se. Rather, it raises the possibility that Cl− binding itself affects K+channel kinetics. Precedent for such an effect exists in corneal epithelia, where extracellular Cl− is necessary for K+ channel opening (29.Rae J.L. Dewey J. Cooper K. Gates P. J. Membr. Biol. 1990; 114: 29-36Crossref PubMed Scopus (14) Google Scholar). The significance of this Cl− dependence in HTC cells is uncertain, but such a mechanism could coordinate the concerted opening of K+ and Cl− channels.The onset of K+ and Cl− current activation occurred within 3–5 min after TNF exposure. These observations implied that channel opening was mediated by an intracellular signaling cascade. Although the precise details of this cascade have not been defined in this study, our data suggest that PKC activation is involved. Consistent with this interpretation, the increase in both K+ and Cl− currents was blocked by (a) chelation of intracellular Ca2+ with EGTA, (b) hydrolysis of intracellular ATP with apyrase, and (c) two structurally unrelated PKC inhibitors with distinct mechanisms of action, chelerythrine and PKC inhibitory peptide. In contrast to findings in neutrophils (22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar), our observations in liver cells indicated that TNF did not increase [Ca2+]i and that TNF-activated currents were not attenuated by inhibition of CaMKII. Thus, mechanisms that couple TNF to channel activation appear to exhibit tissue specificity.PKC has been identified as a mediator for several actions of TNF (30.Wiegmann K. Schutze S. Kampen E. Himmler A. Machleidt T. Kronke M. J. Biol. Chem. 1992; 267: 17997-18001Abstract Full Text PDF PubMed Google Scholar, 31.Gorospe M. Kumar S. Baglioni C. J. Biol. Chem. 1993; 268: 6214-6220Abstract Full Text PDF PubMed Google Scholar, 32.Phelps D.T. Ferro T.J. Higgins P.J. Shankar R. Parker D.M. Johnson A. Am. J. Physiol. 1995; 269: L551-L559PubMed Google Scholar, 33.Wyatt T.A. Ito H. Veys T.J. Spurzem J.R. Am. J. Physiol. 1997; 273: L1007-L1112PubMed Google Scholar, 34.Koller H. Allert N. Oel D. Stoll G. Siebler M. Neuroreport. 1998; 9: 1375-1378Crossref PubMed Scopus (46) Google Scholar), but the PKC isoforms responsible for the K+ and Cl− channel opening in HTC cells remain to be determined. Clues to the types of PKC isoforms involved come from a distinct experimental model of liver cell death, in which the bile acid glycochenodeoxycholic acid produces apoptosis in a PKC-dependent fashion. In this model, glycochenodeoxycholic acid elicits membrane translocation of PKC-α, PKC-δ, and PKC-ε (26.Jones B.A. Rao Y.P. Stravitz R.T. Gores G.J. Am. J. Physiol. 1997; 272: G1109-G1115PubMed Google Scholar). This raises the possibility that one (or more) of these PKC isoforms may be relevant to the actions of TNF described in the present study. In HTC cells, PKC-α appears to mediate the opening of K+ channels in response to oxidants and Cl−channels in response to swelling (27.Wang Y. Sostman A. Roman R. Stribling S. Vigna S. Hannun Y. Raymond J. Fitz J.G. J. Biol. Chem. 1996; 271: 18107-18113Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 28.Roman R.M. Bodily K.O. Wang Y. Raymond J.R Fitz J.G. Hepatology. 1998; 28: 1073-1080Crossref PubMed Scopus (48) Google Scholar). It is thus conceivable that PKC-α may also couple TNF to K+ and Cl−channel activation. This issue is worthy of experimental pursuit.Our findings regarding TNF and its effects on cell death mirror observations made by others in model liver cell systems (11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 12.Kim Y.M. de Vera M.E Watkins S.C Billiar T.R. J. Biol. Chem. 1997; 272: 1402-1411Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). In particular, TNF-mediated cell death required the addition of the transcriptional inhibitor actinomycin D. This implies that TNF activates parallel transcription-dependent pathways (which appear to involve the transcription factor NF-κB, cf. Ref.11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), which serve to prevent cell death. In our hands, liver cell death appeared to occur through both apoptosis and necrosis, given that TNF increased trypan blue uptake (characteristic of necrotic cell death) and the extent of nuclear condensation and/or fragmentation (characteristic of apoptosis) with similar kinetics. Of note, TNF-mediated K+ and Cl− channel activation (within minutes) and cessation of channel activation (by 1 h) occurred much earlier than the onset of cell death (within 4 h). This suggests that these channels occupy early positions in TNF-mediated signaling pathways. Furthermore, our findings raise the possibility that one of these pathways could lead to cell death. Consistent with this interpretation, we have shown that K+and Cl− channel blockers delay the progression to TNF-mediated liver cell death. With this in mind, K+ and Cl− channels may represent attractive therapeutic targets to attenuate liver injury from TNF.It should be emphasized that TNF alone was sufficient to evoke K+ and Cl− channel opening in HTC cells, but cell death required the addition of actinomycin D. This implies that signaling cascades enabled by K+ and Cl−channel opening would not overcome cytoprotective pathways disabled by actinomycin D (see above). In the presence of actinomycin D, K+ and Cl− channel blockade reduced cell death for up to 8 h after TNF exposure, a time in which near maximal cell death had occurred in the absence of channel blockade. However, K+ and Cl− channel blockade did not ultimately prevent TNF-mediated cell death. A possible interpretation is that K+ and Cl− channels participate in the early phases of TNF-mediated signaling pathways that lead to cell death but that later onset, channel-independent pathways are also involved in the death response to TNF. Precedent for this concept exists in biphasic activation of Jun and p38 kinases by TNF, the early phase of which is anti-apoptotic and the later phase of which appears to be linked to apoptosis (35.Roulston A. Reinhard C. Amiri P. Williams L.T. J. Biol. Chem. 1998; 273: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Similarly, in selected instances, the anti-apoptotic protein Bcl-2 may only delay cell death (36.Bissonnette R.P. Echeverri F. Mahboubi A. Green D.R. Nature. 1992; 359: 552-554Crossref PubMed Scopus (922) Google Scholar, 37.Yin D.X. Schimke R.T. Cancer Res. 1995; 55: 4922-4928PubMed Google Scholar), suggesting the existence of parallel pathways to cell death that exhibit distinct temporal characteristics.Although our data support a role for K+ and Cl− channels in TNF-mediated liver cell death, two additional caveats apply. First, Ba2+, quinine, NPPB, and DPC, each of which delayed cell death induced by TNF, may have had effects other than blockade of K+ or Cl−channels. The use of structurally dissimilar agents renders this less likely, but the possibility of nonspecific effects cannot be discounted. Second, the observations reported here may not apply to all mammalian tissues. In particular, in astrocytes, TNF has been shown to inhibit rather than activate K+ currents (34.Koller H. Allert N. Oel D. Stoll G. Siebler M. Neuroreport. 1998; 9: 1375-1378Crossref PubMed Scopus (46) Google Scholar). Thus, the results reported in the present study must be extrapolated with care.An important question is how activation of K+ and Cl− channels contributes to downstream effects of TNF. One intriguing possibility is that K+ and Cl−efflux through conductive pathways leads to liver cell shrinkage. Although speculative, this could have at least two consequences. First, cell shrinkage itself can lead to apoptosis (38.Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 39.Kurz A.K. Schliess F. Haussinger D. Hepatology. 1998; 28: 774-781Crossref PubMed Scopus (48) Google Scholar, 40.Bilney A.J. Murray A.W. FEBS Lett. 1998; 424: 221-224Crossref PubMed Scopus (29) Google Scholar, 41.Chan W.H., Yu, J.S. Yang S.D. J. Cell. Physiol. 1999; 178: 397-408Crossref PubMed Scopus (50) Google Scholar). A second consequence could be reduction of bile formation, achieved through volume-sensitive inhibition of insertion of organic anion transporters into the apical membrane (42.Haussinger D. Saha N. Hallbrucker C. Lang F. Gerok W. Biochem. J. 1993; 291: 355-360Crossref PubMed Scopus (85) Google Scholar, 43.Kubitz R. D'urso D. Keppler D. Haussinger D. Gastroenterology. 1997; 113: 1438-1442Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). This too is consistent with effects of TNF, which reduces both bile flow as well as the abundance of plasma membrane organic anion transporters in hepatocytes (10.Whiting J.F. Green R.M. Rosenbluth A.B Gollan J.L. Hepatology. 1995; 22: 1273-1278PubMed Google Scholar, 16.Moseley R.H. Wang W. Takeda H. Lown K. Shick L. Ananthanarayanan M. Suchy F.J. Am. J. Physiol. 1996; 271: G137-G146PubMed Google Scholar). These areas are worthy of further study and could lead to new insights with respect to TNF action. Tumor necrosis factor α (TNF)1 is an inflammatory cytokine that induces programmed cell death in a variety of tissue types (1.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar). In the liver, TNF has been implicated as a mediator of hepatocellular dysfunction and death following toxic injury, viral hepatitis, and sepsis (2.Czaja M.J. Xu J. Alt E. Gastroenterology. 1995; 108: 1849-1854Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 3.Iimuro Y. Gallucci R.M. Luster M.I. Kono H. Thurman R.G. Hepatology. 1997; 26: 1530-1537Crossref PubMed Scopus (446) Google Scholar, 4.Bird G.L. Sheron N. Goka A.K. Alexander G.J. Williams R.S. Ann. Intern. Med. 1990; 112: 917-920Crossref PubMed Scopus (430) Google Scholar, 5.Mizuhara H. O'Neill E. Seki N. Ogawa T. Kusunoki C. Otsuka K. Satoh S. Niwa M. Senoh H. Fujiwara H. J. Exp. Med. 1994; 179: 1529-1537Crossref PubMed Scopus (493) Google Scholar, 6.Ando K. Moriyama T. Guidotti L.G. Wirth S. Schreiber R.D. Schlicht H.J. Huang S.N. Chisari F.V. J. Exp. Med. 1993; 178: 1541-1554Crossref PubMed Scopus (414) Google Scholar, 7.Ando K. Hiroishi K. Kaneko T. Moriyama T. Muto Y. Kayagaki N. Yagita H. Okumura K. Imawari M. J. Immunol. 1997; 158: 5283-5291PubMed Google Scholar, 8.Jaeschke H. Fisher M.A. Lawson J.A Simmons C.A. Farhood A. Jones D.A. J. Immunol. 1998; 160: 3480-3486PubMed Google Scholar). It is thought that such pathological conditions lead to the release of TNF by hepatic macrophages, with resultant paracrine actions on other liver cells (9.Bradham C.A. Plumpe J. Manns M.P. Brenner D.A. Trautwein C. Am. J. Physiol. 1998; 275: G387-G392PubMed Google Scholar). In liver, TNF exhibits pleiotropic effects, ranging from reduction of bile flow to hepatocellular apoptosis (10.Whiting J.F. Green R.M. Rosenbluth A.B Gollan J.L. Hepatology. 1995; 22: 1273-1278PubMed Google Scholar, 11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 12.Kim Y.M. de Vera M.E Watkins S.C Billiar T.R. J. Biol. Chem. 1997; 272: 1402-1411Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). Experimental evidence supports several mechanisms to account for such effects, including activation of caspases and kinases, generation of free radicals, and down-regulation of membrane organic solute transporters (13.Gross A. Yin X.M. Wang K. Wei M.C. Jockel J. Milliman C. Erdjument-Bromage H. Tempst P. Korsmeyer S.J. J. Biol. Chem. 1999; 274: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar, 14.Ventura J.J. Roncero C. Fabregat I. Benito M. Hepatology. 1999; 29: 849-857Crossref PubMed Scopus (20) Google Scholar, 15.Fernandez-Checa J.C. Kaplowitz N. Garcia-Ruiz C. Colell A. Miranda M. Mari M. Ardite E. Morales A. Am. J. Physiol. 1997; 273: G7-G17Crossref PubMed Google Scholar, 16.Moseley R.H. Wang W. Takeda H. Lown K. Shick L. Ananthanarayanan M. Suchy F.J. Am. J. Physiol. 1996; 271: G137-G146PubMed Google Scholar, 17.Green R.M. Beier D. Gollan J.L. Gastroenterology. 1996; 111: 193-198Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Despite this body of evidence, there remain significant gaps in our knowledge regarding the responsible pathways that couple TNF to liver damage. There is abundant information to suggest that apoptosis is associated with increases in mitochondrial membrane permeability but considerably less is known with respect to the role of the plasma membrane. In a limited number of cell types, increases in plasma membrane permeability to ions represent early responses to apoptotic stimuli. In cultured neurons, serum withdrawal increases voltage-activated K+channel activity and cell death, and in lymphocytes, engagement of the cell surface protein Fas leads to K+ loss, activation of outwardly rectifying Cl− channels, and apoptosis (18.Yu S.P. Yeh C.H. Sensi S.L. Gwag B.J. Canzoniero L.M. Farhangrazi Z.S. Ying H.S. Tian M. Dugan L.L. Choi D.W. Science. 1997; 278: 114-117Crossref PubMed Scopus (532) Google Scholar, 19.Hughes Jr., F.M. Bortner C.D. Purdy G.D. Cidlowski J.A. J. Biol. Chem. 1997; 272: 30567-30576Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 20.Szabo I. Lepple-Wienhues A. Kaba K.N. Zoratti M. Gulbins E. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6169-6174Crossref PubMed Scopus (197) Google Scholar). Conversely, pharmacological blockade of K+ or Cl− channels blunts apoptosis in these experimental models. Thus, K+ and Cl− channels may play important roles in apoptotic processes. Based on its function in inducing apoptosis, there is reason to believe that TNF could influence cell death through activation of ion channels. In support of this, TNF increases K+ currents in selected neurons and activates Cl− currents in neutrophils (21.Houzen H. Kikuchi S. Kanno M. Shinpo K. Tashiro K. J Neurosci. Res. 1997; 50: 990-999Crossref PubMed Scopus (66) Google Scholar,22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar). However, it is unknown whether TNF affects plasma membrane ion channels in other cell types or whether such channels are involved in TNF-mediated cell death. In this study, we have examined the effects of TNF on plasma membrane conductance and death in the model hepatocyte-like cell line HTC. Our results suggest that TNF activates K+ and Cl− channels and that channel activation is an early signal in pathways leading to TNF-mediated liver cell death. DISCUSSIONIn this study, we have examined the role of plasma membrane ion channels in TNF signaling. Our observations suggest that TNF increases membrane permeability to K+ and Cl− in HTC cells through activation of ion channels and that TNF-mediated cell death is delayed by blockade of these channels. Our data are thus consistent with the hypothesis that channel opening is an early event in a TNF-mediated pathway that leads to liver cell death.Two principal findings support the concept that TNF activates K+ and Cl− channels. First, TNF increased membrane currents (I0 and I−80) at potentials corresponding to the opening of K+ and Cl−channels. Second, the TNF-evoked increases in these currents were selectively prevented by either K+ or Cl−removal or by exposure to K+ or Cl− channel blockers, respectively. Our data thus extend observations in neurons, in which TNF activates K+ channels (21.Houzen H. Kikuchi S. Kanno M. Shinpo K. Tashiro K. J Neurosci. Res. 1997; 50: 990-999Crossref PubMed Scopus (66) Google Scholar), and neutrophils, in which TNF activates Cl− channels (22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar), and demonstrate that TNF can activate K+ and Cl− channels in the same cell type.Unexpectedly, the TNF-elicited increase in K+ current (I0) was inhibited by Cl− removal. However, this current was not attenuated by Cl− channel blockers. This suggests that K+ channel opening does not depend on Cl− movement per se. Rather, it raises the possibility that Cl− binding itself affects K+channel kinetics. Precedent for such an effect exists in corneal epithelia, where extracellular Cl− is necessary for K+ channel opening (29.Rae J.L. Dewey J. Cooper K. Gates P. J. Membr. Biol. 1990; 114: 29-36Crossref PubMed Scopus (14) Google Scholar). The significance of this Cl− dependence in HTC cells is uncertain, but such a mechanism could coordinate the concerted opening of K+ and Cl− channels.The onset of K+ and Cl− current activation occurred within 3–5 min after TNF exposure. These observations implied that channel opening was mediated by an intracellular signaling cascade. Although the precise details of this cascade have not been defined in this study, our data suggest that PKC activation is involved. Consistent with this interpretation, the increase in both K+ and Cl− currents was blocked by (a) chelation of intracellular Ca2+ with EGTA, (b) hydrolysis of intracellular ATP with apyrase, and (c) two structurally unrelated PKC inhibitors with distinct mechanisms of action, chelerythrine and PKC inhibitory peptide. In contrast to findings in neutrophils (22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar), our observations in liver cells indicated that TNF did not increase [Ca2+]i and that TNF-activated currents were not attenuated by inhibition of CaMKII. Thus, mechanisms that couple TNF to channel activation appear to exhibit tissue specificity.PKC has been identified as a mediator for several actions of TNF (30.Wiegmann K. Schutze S. Kampen E. Himmler A. Machleidt T. Kronke M. J. Biol. Chem. 1992; 267: 17997-18001Abstract Full Text PDF PubMed Google Scholar, 31.Gorospe M. Kumar S. Baglioni C. J. Biol. Chem. 1993; 268: 6214-6220Abstract Full Text PDF PubMed Google Scholar, 32.Phelps D.T. Ferro T.J. Higgins P.J. Shankar R. Parker D.M. Johnson A. Am. J. Physiol. 1995; 269: L551-L559PubMed Google Scholar, 33.Wyatt T.A. Ito H. Veys T.J. Spurzem J.R. Am. J. Physiol. 1997; 273: L1007-L1112PubMed Google Scholar, 34.Koller H. Allert N. Oel D. Stoll G. Siebler M. Neuroreport. 1998; 9: 1375-1378Crossref PubMed Scopus (46) Google Scholar), but the PKC isoforms responsible for the K+ and Cl− channel opening in HTC cells remain to be determined. Clues to the types of PKC isoforms involved come from a distinct experimental model of liver cell death, in which the bile acid glycochenodeoxycholic acid produces apoptosis in a PKC-dependent fashion. In this model, glycochenodeoxycholic acid elicits membrane translocation of PKC-α, PKC-δ, and PKC-ε (26.Jones B.A. Rao Y.P. Stravitz R.T. Gores G.J. Am. J. Physiol. 1997; 272: G1109-G1115PubMed Google Scholar). This raises the possibility that one (or more) of these PKC isoforms may be relevant to the actions of TNF described in the present study. In HTC cells, PKC-α appears to mediate the opening of K+ channels in response to oxidants and Cl−channels in response to swelling (27.Wang Y. Sostman A. Roman R. Stribling S. Vigna S. Hannun Y. Raymond J. Fitz J.G. J. Biol. Chem. 1996; 271: 18107-18113Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 28.Roman R.M. Bodily K.O. Wang Y. Raymond J.R Fitz J.G. Hepatology. 1998; 28: 1073-1080Crossref PubMed Scopus (48) Google Scholar). It is thus conceivable that PKC-α may also couple TNF to K+ and Cl−channel activation. This issue is worthy of experimental pursuit.Our findings regarding TNF and its effects on cell death mirror observations made by others in model liver cell systems (11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 12.Kim Y.M. de Vera M.E Watkins S.C Billiar T.R. J. Biol. Chem. 1997; 272: 1402-1411Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). In particular, TNF-mediated cell death required the addition of the transcriptional inhibitor actinomycin D. This implies that TNF activates parallel transcription-dependent pathways (which appear to involve the transcription factor NF-κB, cf. Ref.11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), which serve to prevent cell death. In our hands, liver cell death appeared to occur through both apoptosis and necrosis, given that TNF increased trypan blue uptake (characteristic of necrotic cell death) and the extent of nuclear condensation and/or fragmentation (characteristic of apoptosis) with similar kinetics. Of note, TNF-mediated K+ and Cl− channel activation (within minutes) and cessation of channel activation (by 1 h) occurred much earlier than the onset of cell death (within 4 h). This suggests that these channels occupy early positions in TNF-mediated signaling pathways. Furthermore, our findings raise the possibility that one of these pathways could lead to cell death. Consistent with this interpretation, we have shown that K+and Cl− channel blockers delay the progression to TNF-mediated liver cell death. With this in mind, K+ and Cl− channels may represent attractive therapeutic targets to attenuate liver injury from TNF.It should be emphasized that TNF alone was sufficient to evoke K+ and Cl− channel opening in HTC cells, but cell death required the addition of actinomycin D. This implies that signaling cascades enabled by K+ and Cl−channel opening would not overcome cytoprotective pathways disabled by actinomycin D (see above). In the presence of actinomycin D, K+ and Cl− channel blockade reduced cell death for up to 8 h after TNF exposure, a time in which near maximal cell death had occurred in the absence of channel blockade. However, K+ and Cl− channel blockade did not ultimately prevent TNF-mediated cell death. A possible interpretation is that K+ and Cl− channels participate in the early phases of TNF-mediated signaling pathways that lead to cell death but that later onset, channel-independent pathways are also involved in the death response to TNF. Precedent for this concept exists in biphasic activation of Jun and p38 kinases by TNF, the early phase of which is anti-apoptotic and the later phase of which appears to be linked to apoptosis (35.Roulston A. Reinhard C. Amiri P. Williams L.T. J. Biol. Chem. 1998; 273: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Similarly, in selected instances, the anti-apoptotic protein Bcl-2 may only delay cell death (36.Bissonnette R.P. Echeverri F. Mahboubi A. Green D.R. Nature. 1992; 359: 552-554Crossref PubMed Scopus (922) Google Scholar, 37.Yin D.X. Schimke R.T. Cancer Res. 1995; 55: 4922-4928PubMed Google Scholar), suggesting the existence of parallel pathways to cell death that exhibit distinct temporal characteristics.Although our data support a role for K+ and Cl− channels in TNF-mediated liver cell death, two additional caveats apply. First, Ba2+, quinine, NPPB, and DPC, each of which delayed cell death induced by TNF, may have had effects other than blockade of K+ or Cl−channels. The use of structurally dissimilar agents renders this less likely, but the possibility of nonspecific effects cannot be discounted. Second, the observations reported here may not apply to all mammalian tissues. In particular, in astrocytes, TNF has been shown to inhibit rather than activate K+ currents (34.Koller H. Allert N. Oel D. Stoll G. Siebler M. Neuroreport. 1998; 9: 1375-1378Crossref PubMed Scopus (46) Google Scholar). Thus, the results reported in the present study must be extrapolated with care.An important question is how activation of K+ and Cl− channels contributes to downstream effects of TNF. One intriguing possibility is that K+ and Cl−efflux through conductive pathways leads to liver cell shrinkage. Although speculative, this could have at least two consequences. First, cell shrinkage itself can lead to apoptosis (38.Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 39.Kurz A.K. Schliess F. Haussinger D. Hepatology. 1998; 28: 774-781Crossref PubMed Scopus (48) Google Scholar, 40.Bilney A.J. Murray A.W. FEBS Lett. 1998; 424: 221-224Crossref PubMed Scopus (29) Google Scholar, 41.Chan W.H., Yu, J.S. Yang S.D. J. Cell. Physiol. 1999; 178: 397-408Crossref PubMed Scopus (50) Google Scholar). A second consequence could be reduction of bile formation, achieved through volume-sensitive inhibition of insertion of organic anion transporters into the apical membrane (42.Haussinger D. Saha N. Hallbrucker C. Lang F. Gerok W. Biochem. J. 1993; 291: 355-360Crossref PubMed Scopus (85) Google Scholar, 43.Kubitz R. D'urso D. Keppler D. Haussinger D. Gastroenterology. 1997; 113: 1438-1442Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). This too is consistent with effects of TNF, which reduces both bile flow as well as the abundance of plasma membrane organic anion transporters in hepatocytes (10.Whiting J.F. Green R.M. Rosenbluth A.B Gollan J.L. Hepatology. 1995; 22: 1273-1278PubMed Google Scholar, 16.Moseley R.H. Wang W. Takeda H. Lown K. Shick L. Ananthanarayanan M. Suchy F.J. Am. J. Physiol. 1996; 271: G137-G146PubMed Google Scholar). These areas are worthy of further study and could lead to new insights with respect to TNF action. In this study, we have examined the role of plasma membrane ion channels in TNF signaling. Our observations suggest that TNF increases membrane permeability to K+ and Cl− in HTC cells through activation of ion channels and that TNF-mediated cell death is delayed by blockade of these channels. Our data are thus consistent with the hypothesis that channel opening is an early event in a TNF-mediated pathway that leads to liver cell death. Two principal findings support the concept that TNF activates K+ and Cl− channels. First, TNF increased membrane currents (I0 and I−80) at potentials corresponding to the opening of K+ and Cl−channels. Second, the TNF-evoked increases in these currents were selectively prevented by either K+ or Cl−removal or by exposure to K+ or Cl− channel blockers, respectively. Our data thus extend observations in neurons, in which TNF activates K+ channels (21.Houzen H. Kikuchi S. Kanno M. Shinpo K. Tashiro K. J Neurosci. Res. 1997; 50: 990-999Crossref PubMed Scopus (66) Google Scholar), and neutrophils, in which TNF activates Cl− channels (22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar), and demonstrate that TNF can activate K+ and Cl− channels in the same cell type. Unexpectedly, the TNF-elicited increase in K+ current (I0) was inhibited by Cl− removal. However, this current was not attenuated by Cl− channel blockers. This suggests that K+ channel opening does not depend on Cl− movement per se. Rather, it raises the possibility that Cl− binding itself affects K+channel kinetics. Precedent for such an effect exists in corneal epithelia, where extracellular Cl− is necessary for K+ channel opening (29.Rae J.L. Dewey J. Cooper K. Gates P. J. Membr. Biol. 1990; 114: 29-36Crossref PubMed Scopus (14) Google Scholar). The significance of this Cl− dependence in HTC cells is uncertain, but such a mechanism could coordinate the concerted opening of K+ and Cl− channels. The onset of K+ and Cl− current activation occurred within 3–5 min after TNF exposure. These observations implied that channel opening was mediated by an intracellular signaling cascade. Although the precise details of this cascade have not been defined in this study, our data suggest that PKC activation is involved. Consistent with this interpretation, the increase in both K+ and Cl− currents was blocked by (a) chelation of intracellular Ca2+ with EGTA, (b) hydrolysis of intracellular ATP with apyrase, and (c) two structurally unrelated PKC inhibitors with distinct mechanisms of action, chelerythrine and PKC inhibitory peptide. In contrast to findings in neutrophils (22.Schumann M.A. Gardner P. Raffin T.A. J. Biol. Chem. 1993; 268: 2134-2140Abstract Full Text PDF PubMed Google Scholar), our observations in liver cells indicated that TNF did not increase [Ca2+]i and that TNF-activated currents were not attenuated by inhibition of CaMKII. Thus, mechanisms that couple TNF to channel activation appear to exhibit tissue specificity. PKC has been identified as a mediator for several actions of TNF (30.Wiegmann K. Schutze S. Kampen E. Himmler A. Machleidt T. Kronke M. J. Biol. Chem. 1992; 267: 17997-18001Abstract Full Text PDF PubMed Google Scholar, 31.Gorospe M. Kumar S. Baglioni C. J. Biol. Chem. 1993; 268: 6214-6220Abstract Full Text PDF PubMed Google Scholar, 32.Phelps D.T. Ferro T.J. Higgins P.J. Shankar R. Parker D.M. Johnson A. Am. J. Physiol. 1995; 269: L551-L559PubMed Google Scholar, 33.Wyatt T.A. Ito H. Veys T.J. Spurzem J.R. Am. J. Physiol. 1997; 273: L1007-L1112PubMed Google Scholar, 34.Koller H. Allert N. Oel D. Stoll G. Siebler M. Neuroreport. 1998; 9: 1375-1378Crossref PubMed Scopus (46) Google Scholar), but the PKC isoforms responsible for the K+ and Cl− channel opening in HTC cells remain to be determined. Clues to the types of PKC isoforms involved come from a distinct experimental model of liver cell death, in which the bile acid glycochenodeoxycholic acid produces apoptosis in a PKC-dependent fashion. In this model, glycochenodeoxycholic acid elicits membrane translocation of PKC-α, PKC-δ, and PKC-ε (26.Jones B.A. Rao Y.P. Stravitz R.T. Gores G.J. Am. J. Physiol. 1997; 272: G1109-G1115PubMed Google Scholar). This raises the possibility that one (or more) of these PKC isoforms may be relevant to the actions of TNF described in the present study. In HTC cells, PKC-α appears to mediate the opening of K+ channels in response to oxidants and Cl−channels in response to swelling (27.Wang Y. Sostman A. Roman R. Stribling S. Vigna S. Hannun Y. Raymond J. Fitz J.G. J. Biol. Chem. 1996; 271: 18107-18113Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 28.Roman R.M. Bodily K.O. Wang Y. Raymond J.R Fitz J.G. Hepatology. 1998; 28: 1073-1080Crossref PubMed Scopus (48) Google Scholar). It is thus conceivable that PKC-α may also couple TNF to K+ and Cl−channel activation. This issue is worthy of experimental pursuit. Our findings regarding TNF and its effects on cell death mirror observations made by others in model liver cell systems (11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 12.Kim Y.M. de Vera M.E Watkins S.C Billiar T.R. J. Biol. Chem. 1997; 272: 1402-1411Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). In particular, TNF-mediated cell death required the addition of the transcriptional inhibitor actinomycin D. This implies that TNF activates parallel transcription-dependent pathways (which appear to involve the transcription factor NF-κB, cf. Ref.11.Xu Y. Bialik S. Jones B.E Iimuro Y. Kitsis R.N Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), which serve to prevent cell death. In our hands, liver cell death appeared to occur through both apoptosis and necrosis, given that TNF increased trypan blue uptake (characteristic of necrotic cell death) and the extent of nuclear condensation and/or fragmentation (characteristic of apoptosis) with similar kinetics. Of note, TNF-mediated K+ and Cl− channel activation (within minutes) and cessation of channel activation (by 1 h) occurred much earlier than the onset of cell death (within 4 h). This suggests that these channels occupy early positions in TNF-mediated signaling pathways. Furthermore, our findings raise the possibility that one of these pathways could lead to cell death. Consistent with this interpretation, we have shown that K+and Cl− channel blockers delay the progression to TNF-mediated liver cell death. With this in mind, K+ and Cl− channels may represent attractive therapeutic targets to attenuate liver injury from TNF. It should be emphasized that TNF alone was sufficient to evoke K+ and Cl− channel opening in HTC cells, but cell death required the addition of actinomycin D. This implies that signaling cascades enabled by K+ and Cl−channel opening would not overcome cytoprotective pathways disabled by actinomycin D (see above). In the presence of actinomycin D, K+ and Cl− channel blockade reduced cell death for up to 8 h after TNF exposure, a time in which near maximal cell death had occurred in the absence of channel blockade. However, K+ and Cl− channel blockade did not ultimately prevent TNF-mediated cell death. A possible interpretation is that K+ and Cl− channels participate in the early phases of TNF-mediated signaling pathways that lead to cell death but that later onset, channel-independent pathways are also involved in the death response to TNF. Precedent for this concept exists in biphasic activation of Jun and p38 kinases by TNF, the early phase of which is anti-apoptotic and the later phase of which appears to be linked to apoptosis (35.Roulston A. Reinhard C. Amiri P. Williams L.T. J. Biol. Chem. 1998; 273: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Similarly, in selected instances, the anti-apoptotic protein Bcl-2 may only delay cell death (36.Bissonnette R.P. Echeverri F. Mahboubi A. Green D.R. Nature. 1992; 359: 552-554Crossref PubMed Scopus (922) Google Scholar, 37.Yin D.X. Schimke R.T. Cancer Res. 1995; 55: 4922-4928PubMed Google Scholar), suggesting the existence of parallel pathways to cell death that exhibit distinct temporal characteristics. Although our data support a role for K+ and Cl− channels in TNF-mediated liver cell death, two additional caveats apply. First, Ba2+, quinine, NPPB, and DPC, each of which delayed cell death induced by TNF, may have had effects other than blockade of K+ or Cl−channels. The use of structurally dissimilar agents renders this less likely, but the possibility of nonspecific effects cannot be discounted. Second, the observations reported here may not apply to all mammalian tissues. In particular, in astrocytes, TNF has been shown to inhibit rather than activate K+ currents (34.Koller H. Allert N. Oel D. Stoll G. Siebler M. Neuroreport. 1998; 9: 1375-1378Crossref PubMed Scopus (46) Google Scholar). Thus, the results reported in the present study must be extrapolated with care. An important question is how activation of K+ and Cl− channels contributes to downstream effects of TNF. One intriguing possibility is that K+ and Cl−efflux through conductive pathways leads to liver cell shrinkage. Although speculative, this could have at least two consequences. First, cell shrinkage itself can lead to apoptosis (38.Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 39.Kurz A.K. Schliess F. Haussinger D. Hepatology. 1998; 28: 774-781Crossref PubMed Scopus (48) Google Scholar, 40.Bilney A.J. Murray A.W. FEBS Lett. 1998; 424: 221-224Crossref PubMed Scopus (29) Google Scholar, 41.Chan W.H., Yu, J.S. Yang S.D. J. Cell. Physiol. 1999; 178: 397-408Crossref PubMed Scopus (50) Google Scholar). A second consequence could be reduction of bile formation, achieved through volume-sensitive inhibition of insertion of organic anion transporters into the apical membrane (42.Haussinger D. Saha N. Hallbrucker C. Lang F. Gerok W. Biochem. J. 1993; 291: 355-360Crossref PubMed Scopus (85) Google Scholar, 43.Kubitz R. D'urso D. Keppler D. Haussinger D. Gastroenterology. 1997; 113: 1438-1442Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). This too is consistent with effects of TNF, which reduces both bile flow as well as the abundance of plasma membrane organic anion transporters in hepatocytes (10.Whiting J.F. Green R.M. Rosenbluth A.B Gollan J.L. Hepatology. 1995; 22: 1273-1278PubMed Google Scholar, 16.Moseley R.H. Wang W. Takeda H. Lown K. Shick L. Ananthanarayanan M. Suchy F.J. Am. J. Physiol. 1996; 271: G137-G146PubMed Google Scholar). These areas are worthy of further study and could lead to new insights with respect to TNF action." @default.
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W1998248849 title "Activation of Potassium and Chloride Channels by Tumor Necrosis Factor α" @default.
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