Journal of Biological Chemistry · 1996 · 33 citations · 33 references
Life SciencesPharmacologyPhysiologyCell SignalingIon Channel BiophysicsSignal TransductionIon Channel PharmacologyIon Channel PhysiologyIon Channel RegulationIon Channel SignalingIon ChannelsPotassium Channel BiophysicsCalcium-activated Potassium ChannelsChannel Gating MechanismsIon Channel ActivationPotassium Channel ConductancePotassium Channel FunctionPotassium Channel GatingPotassium Channel RegulationStress-activated Protein Kinases
These studies of a model liver cell line evaluate the mechanisms responsible for regulated release of K+ ions during metabolic stress. Metabolic inhibition of HTC hepatoma cells by exposure to 2,4-dinitrophenol (50 µM) and 2-deoxy-D-glucose (10 mM) stimulated outward currents carried by K+ of 974 ± 75 pA at 0 mV (n = 20, p < 0.001). Currents were inhibited by chelation of intracellular Ca2+ or exposure to apamin (50 nM), an inhibitor of SKCa channels. In cell-attached recordings from intact cells, removal of metabolic substrates (25/28 cells) or exposure to metabolic inhibitors (32/40 cells) opened K+-selective channels with a conductance of 6.5 ± 0.2 pS. Channels had an open probability of 0.31 ± 0.08 and opened in bursts averaging 3.55 ± 0.27 ms in duration (n = 6). Metabolic stress was associated with rapid translocation of the α isoform of protein kinase C (PKCα) from cytosol to membrane; and down-regulation of PKCα by phorbol esters or exposure to the PKC inhibitor chelerythrine (10 µM) each inhibited currents. Moreover, intracellular perfusion with purified PKCα activated currents in a Ca2+- and concentration-dependent manner. These findings indicate that metabolic stress leads to opening of apamin-sensitive SKCa channels in hepatoma cells through a Ca2+- and PKC-dependent mechanism and suggest that PKCα may be selectively involved in the response. This mechanism functionally couples the metabolic state of cells to membrane K+ permeability and represents a potential target for modification of liver injury associated with ischemia and preservation. These studies of a model liver cell line evaluate the mechanisms responsible for regulated release of K+ ions during metabolic stress. Metabolic inhibition of HTC hepatoma cells by exposure to 2,4-dinitrophenol (50 µM) and 2-deoxy-D-glucose (10 mM) stimulated outward currents carried by K+ of 974 ± 75 pA at 0 mV (n = 20, p < 0.001). Currents were inhibited by chelation of intracellular Ca2+ or exposure to apamin (50 nM), an inhibitor of SKCa channels. In cell-attached recordings from intact cells, removal of metabolic substrates (25/28 cells) or exposure to metabolic inhibitors (32/40 cells) opened K+-selective channels with a conductance of 6.5 ± 0.2 pS. Channels had an open probability of 0.31 ± 0.08 and opened in bursts averaging 3.55 ± 0.27 ms in duration (n = 6). Metabolic stress was associated with rapid translocation of the α isoform of protein kinase C (PKCα) from cytosol to membrane; and down-regulation of PKCα by phorbol esters or exposure to the PKC inhibitor chelerythrine (10 µM) each inhibited currents. Moreover, intracellular perfusion with purified PKCα activated currents in a Ca2+- and concentration-dependent manner. These findings indicate that metabolic stress leads to opening of apamin-sensitive SKCa channels in hepatoma cells through a Ca2+- and PKC-dependent mechanism and suggest that PKCα may be selectively involved in the response. This mechanism functionally couples the metabolic state of cells to membrane K+ permeability and represents a potential target for modification of liver injury associated with ischemia and preservation. INTRODUCTIONK+ efflux through ion channels represents a key point for regulation of liver cell transport and metabolism. Under physiologic conditions, opening of K+ channels by glucagon and closure by insulin modulates many liver cell functions including solute uptake, gluconeogenesis, and intracellular pH regulation through effects on membrane potential difference (1Friedmann N. Dambach G. Biochim. Biophys. Acta. 1980; 596: 180-185Google Scholar, 2Fitz J.G. Lidofsky S.D. Xie M.H. Scharschmidt B.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4197-4201Google Scholar). However, these regulatory pathways are disrupted by metabolic stress associated with exposure to toxins including ethanol (3Matsumura T. Thurman R.G. Eur. J. Biochem. 1984; 140: 229-234Google Scholar) or by ischemia related to hypoperfusion or organ preservation which can result in liver cell injury and necrosis (4Rosser B.G. Gores G. Gastroenterology. 1995; 108: 252-275Google Scholar). Many of the effects of ischemia result from hypoxia which inhibits oxidative phosphorylation and depletes cellular ATP stores. Pharmacologic agents including 2,4-dinitrophenol (DNP) 1The abbreviations used are: DNP2,4-dinitrophenol2-DG2-deoxy-D-glucosePKCprotein kinase CPMAphorbol 12-myristate 13acetateIP3inositol 1,4,5-triphosphateLDHlactate dehydrogenasePBSphosphate-buffered salineFCCPcarbonyl cyanide p-tri fluoromethoxyphenylhydrazone. which uncouples oxidative metabolism and 2-deoxy-D-glucose (2-DG) which impairs glycolysis have been useful models of ATP depletion because in liver cells they produce biochemical changes similar to those caused by hypoxia (5Kehrer J.P. Jones D.P. Lemasters J.J. Farber J.L. Jaeschke H. Toxicol. Appl. Pharmacol. 1990; 106: 165-178Google Scholar).At the cellular level, one of the earliest effects of oxidative stress is dissipation of transmembrane cation gradients (6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar) and sustained release of K+ ions (7Hallbrucker C. Ritter M. Lang F. Gerok W. Haussinger D. Eur. J. Biochem. 1993; 211: 449-458Google Scholar). In isolated livers, oxidative stress stimulates K+ efflux, increasing the concentration of K+ in perfusate from 5.6 to 13.1 mM (8Mets B. Hickman R. Allin R. van Dyk J. Lotz Z. Hepatology. 1993; 17: 668-676Google Scholar). Efflux of K+ appears early, preceding release of aminotransferases, and correlates closely with the fall in ATP levels (8Mets B. Hickman R. Allin R. van Dyk J. Lotz Z. Hepatology. 1993; 17: 668-676Google Scholar). Net loss of K+ ions is related in part to inhibition of Na+/K+ pump activity (4Rosser B.G. Gores G. Gastroenterology. 1995; 108: 252-275Google Scholar, 6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar). However, studies of other cell types have identified an additional and quantitatively important pathway for hypoxia-induced K+ efflux mediated by opening of K+ channels.In cardiovascular tissues, K+ channel opening represents an important adaptive response to stress since membrane hyperpolarization results in vasodilation and restoration of blood flow (9Silberberg S.D. van Breemen C. Pfluegers Arch. Eur. J. Physiol. 1992; 420: 118-120Google Scholar). Two modes of regulation have been identified, including opening of glibenclamide-sensitive KATP channels where ATP itself is responsible for channel gating (9Silberberg S.D. van Breemen C. Pfluegers Arch. Eur. J. Physiol. 1992; 420: 118-120Google Scholar, 10Komaru T. Lamping K.G. Eastham C.L. Dellsperger K.C. Circ. Res. 1991; 69: 1146-1151Google Scholar, 11Nichols C.G. Lederer W.J. Am. J. Physiol. 1991; 261: H1675-H1686Google Scholar) and opening of Ca2+-sensitive K+ channels in tissues where metabolic inhibition increases intracellular Ca2+ concentration ([Ca2+]i) (12Miller A.L. Morales E. LeBlanc N.R. Cole W.C. Am. J. Physiol. 1993; 265: H2184-H2195Google Scholar, 13Groschner K. Silberberg S.D. Gelband C.H. van Breemen C. Pfluegers Arch. Eur. J. Physiol. 1991; 417: 517-522Google Scholar). It is notable that metabolic inhibition releases Ca2+ from intracellular stores of liver cells (6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar, 14Lynch C.J. Deth R.C. Pharmacology. 1984; 28: 74-85Google Scholar, 15Tohkin M. Yoshimatsu N. Matsubara T. Jpn. J. Pharmacol. 1988; 46: 61-69Google Scholar), and the resulting increase in [Ca2+]i is characteristic of many forms of liver cell injury (4Rosser B.G. Gores G. Gastroenterology. 1995; 108: 252-275Google Scholar).The purpose of the current studies was to evaluate the relationship between cellular metabolism and membrane K+ permeability in HTC cells, a model liver cell line. Different models of metabolic inhibition increased K+ conductance 30-fold or more through activation of apamin-sensitive SKCa channels. Channel opening appears to occur through a mechanism distinct from cardiovascular tissues and involves translocation of protein kinase Cα (PKCα) from cytosol to membrane. Moreover, intracellular perfusion with purified PKCα activates currents in the absence of metabolic inhibition, suggesting that the α isoform of PKC may be selectively involved in channel opening.DISCUSSIONK+ efflux is an early and characteristic feature of liver cell injury that precedes loss of cell viability (4Rosser B.G. Gores G. Gastroenterology. 1995; 108: 252-275Google Scholar, 7Hallbrucker C. Ritter M. Lang F. Gerok W. Haussinger D. Eur. J. Biochem. 1993; 211: 449-458Google Scholar, 8Mets B. Hickman R. Allin R. van Dyk J. Lotz Z. Hepatology. 1993; 17: 668-676Google Scholar). The principal findings in this model cell line are that K+ efflux is regulated through a Ca2+- and PKC-dependent mechanism and involves opening of apamin-sensitive SKCa channels. If similar mechanisms are operative in primary cells, this represents a potent and previously unrecognized pathway for coupling the metabolic state of cells to changes in membrane K+ permeability.Previous studies indicate that many forms of metabolic stress release Ca2+ from intracellular stores of liver cells and cell lines (6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar, 14Lynch C.J. Deth R.C. Pharmacology. 1984; 28: 74-85Google Scholar, 15Tohkin M. Yoshimatsu N. Matsubara T. Jpn. J. Pharmacol. 1988; 46: 61-69Google Scholar). Ca2+ release in these studies occurred through a mechanism independent of IP3. While the intracellular signals involved in Ca2+ release are not known, these results are consistent with previous studies of liver cells where Ca2+ stores released by DNP are different from those released by epinephrine (15Tohkin M. Yoshimatsu N. Matsubara T. Jpn. J. Pharmacol. 1988; 46: 61-69Google Scholar). Ca2+ release appears to be important for activation of IK since, in whole-cell recordings, buffering intracellular Ca2+ to levels below 50 nM prevented activation by DNP. A role for [Ca2+]i is also supported by results in intact cells where removal of glycolytic substrates produced an oscillatory pattern of opening that was Ca2+-dependent. Channel opening in intact cells was rapidly reversed on return to glucose-containing solutions and occurred in the absence of any toxins. These different maneuvers, glucose removal versus mitochondrial inhibition, are not equivalent in their actions or efficacy. However, they have common effects on ATP stores and produce biochemical changes in liver cells similar to those caused by hypoxia (5Kehrer J.P. Jones D.P. Lemasters J.J. Farber J.L. Jaeschke H. Toxicol. Appl. Pharmacol. 1990; 106: 165-178Google Scholar).Several observations suggest that HTC cells would be anticipated to reflect the behavior of primary hepatocytes. First, the relative abundance of PKC isoforms in HTC cells closely resembles that of hepatocytes, with PKC-α as the primary isoform with strong dependences on calcium, phospholipid, and diacylglycerol; and PKC-δ as the primary calcium-independent isoform (27Wetsel W.C. Khan W.A. Merchenthaler I. Rivera H. Halpern A.E. Phung H.M. Negro-Vilar A. Hannun Y.A. J. Cell Biol. 1992; 117: 121-133Google Scholar). Second, previous studies support similarities between the electrical properties of HTC cells and those of primary hepatocytes (16Lidofsky S.D. Xie M.H. Sostman A. Scharschmidt B.F. Fitz J.G. J. Biol. Chem. 1993; 268: 14632-14636Google Scholar, 18Lidofsky S.D. Proc. Natl. Acad. Sci. U. S. A. 1995; (in press)Google Scholar). Third, HTC cells express insulin receptors (34Heaton J.H. Krett N.L. Alvarez J.M. Gelehrter T.D. Romanus J.A. Rechler M.M. J. Biol. Chem. 1984; 259: 2396-2402Google Scholar) and metabolic pathways for chylomicrons, low density lipoprotein, and very low density lipoprotein that are also similar to primary cells (35Barnard G.F. Erickson S.K. Cooper A.D. J. Clin. Invest. 1984; 74: 173-184Google Scholar). However, there are comparatively few electrophysiologic studies of hepatic cells and no previous information regarding the mechanisms that couple cell metabolism to ion channel activity. Consequently, the limitations inherent in the use of cell lines are acknowledged.While mobilization of cytosolic Ca2+ is necessary for current activation, increasing the Ca2+ concentration of the intracellular solution to 300 nM failed to activate IK, suggesting that other factors are involved as well. Several observations support an important role for PKC. First, addition of ATP to the pipette solution enhanced rather than inhibited current activation by DNP/2-DG. This indicates that ATP depletion per se is not responsible for channel activation and argues against a role for KATP channels since increasing intracellular ATP would be expected to close rather than open these channels. Second, activation of IK was inhibited by the PKC inhibitor chelerythrine and by prolonged exposure to phorbol esters. The concentration of chelerythrine of 10 µM used for these studies is above the Ki for PKC of ∼1 µM but below that required for inhibition of protein kinase A (∼160 µM) or calmodulin-dependent kinase (>100 µM) (36Herbert J.M. Augereau J.M. Gleye J. Maffrand J.P. Biochem. Biophys. Res. Commun. 1990; 172: 993-999Google Scholar), suggesting a selective effect. The actual intracellular concentration of chelerythrine is likely to be lower because of the time required for diffusion out of the pipette and into the cell (37Worrell R.T. Frizzell R.A. Am. J. Physiol. 1991; 260: C877-C882Google Scholar). While the potential for nonspecific effects is acknowledged, in aggregate the studies are most consistent with channel activation through a PKC-dependent pathway.The PKC family of protein-serine kinases has been implicated in regulation of a broad range of cellular functions. Generally, activation involves receptor stimulation, hydrolysis of phosphatidylinositol bisphosphate, and release of diacylglycerol and IP3. In the current studies, however, Ca2+ mobilization and current activation do not appear to involve receptor binding or IP3 generation. However, indirect observations support an important role for PKCα in channel opening. PKCα is the dominant Ca2+-sensitive isoform in HTC cells, and down-regulation of PKCα by phorbol esters prevented activation of IK during metabolic stress. Moreover, exposure of quiescent cells to DNP/2-DG caused translocation of PKCα to the plasma membrane, a measure of activation, in the absence of receptor agonists and was sustained for the 30-min study period.The role of PKCα was evaluated more directly by intracellular perfusion with exogenous PKCα which activated IK in the absence of toxins, consistent with the concept that current activation represents a physiologic response to specific signaling events, and not toxic or nonspecific effects of the different inhibitors. The effects of PKCα were Ca2+-dependent and were sustained for up to 20 min in individual cells. The total activity of the PKCα-containing solutions used for intracellular perfusion ranged between ∼1990 and ∼4969 pmol of PO4·min−1 per µl. However, the actual activity inside the cell in the presence of phosphatases and other regulatory factors cannot be assessed. It is notable that the activation of K+ currents was followed by appearance of inward currents presumably carried by Cl− ions, suggesting a possible role for PKCα in regulation of other channel types as well.The most direct interpretation of these studies is that PKCα stimulates IK by direct phosphorylation of apamin-sensitive SKCa channels (22Fitz J.G. Sostman A. Middleton J.P. Am. J. Physiol. 1994; 266: G677-G684Google Scholar, 38Wang W. Giebisch G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9722-9725Google Scholar, 39Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Google Scholar). However, channels could not be studied in excised patches due to rapid run down within 10-20 s of excision. Consequently, the involvement of an intermediate regulatory phosphoprotein cannot be excluded. If so, Ca2+- and PKC-dependent channel opening could be sustained as long as ATP is present in levels sufficient to support phosphorylation, but over longer periods channel open probability would be expected to decrease as cellular ATP stores fall below a critical level.The physiologic implications of K+ channel opening during periods of metabolic stress in these cells is not known. By analogy with vascular tissues where K+ channels represent a protective adaptive mechanism, opening of K+ channels in liver cells could be beneficial in the early stages of injury since membrane hyperpolarization would stimulate electrogenic uptake of substrates important for regeneration of cellular ATP stores (40Wondergem R. Harder D.R. J. Cell. Physiol. 1980; 104: 53-60Google Scholar) and would inhibit Na+/HCO−3 cotransport and electrogenic influx of HCO−3 ions (2Fitz J.G. Lidofsky S.D. Xie M.H. Scharschmidt B.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4197-4201Google Scholar). Inhibition of HCO−3 transport would contribute to the intracellular acidification that accompanies hypoxia (6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar) and is thought to minimize or prevent cell injury (41Currin R.T. Gores G. Thurman R.G. Lemasters J.J. FASEB J. 1991; 5: 207-210Google Scholar). In addition, K+ efflux is necessary for recovery from cell swelling (42Howard L.D. Wondergem R. J. Membr. Biol. 1987; 100: 53-61Google Scholar, 43Wang K. Wondergem R. Am. J. Physiol. 1992; 263: G566-G572Google Scholar). Over longer periods, however, sustained release of K+ in the setting of Na+/K+ pump inhibition would dissipate transmembrane cation gradients. In view of the fundamental importance of K+ channels to different liver cell functions, it is attractive to speculate that SKCa channels and PKCα represent potential targets for pharmacologic modification of the cellular response to injury. INTRODUCTIONK+ efflux through ion channels represents a key point for regulation of liver cell transport and metabolism. Under physiologic conditions, opening of K+ channels by glucagon and closure by insulin modulates many liver cell functions including solute uptake, gluconeogenesis, and intracellular pH regulation through effects on membrane potential difference (1Friedmann N. Dambach G. Biochim. Biophys. Acta. 1980; 596: 180-185Google Scholar, 2Fitz J.G. Lidofsky S.D. Xie M.H. Scharschmidt B.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4197-4201Google Scholar). However, these regulatory pathways are disrupted by metabolic stress associated with exposure to toxins including ethanol (3Matsumura T. Thurman R.G. Eur. J. Biochem. 1984; 140: 229-234Google Scholar) or by ischemia related to hypoperfusion or organ preservation which can result in liver cell injury and necrosis (4Rosser B.G. Gores G. Gastroenterology. 1995; 108: 252-275Google Scholar). Many of the effects of ischemia result from hypoxia which inhibits oxidative phosphorylation and depletes cellular ATP stores. Pharmacologic agents including 2,4-dinitrophenol (DNP) 1The abbreviations used are: DNP2,4-dinitrophenol2-DG2-deoxy-D-glucosePKCprotein kinase CPMAphorbol 12-myristate 13acetateIP3inositol 1,4,5-triphosphateLDHlactate dehydrogenasePBSphosphate-buffered salineFCCPcarbonyl cyanide p-tri fluoromethoxyphenylhydrazone. which uncouples oxidative metabolism and 2-deoxy-D-glucose (2-DG) which impairs glycolysis have been useful models of ATP depletion because in liver cells they produce biochemical changes similar to those caused by hypoxia (5Kehrer J.P. Jones D.P. Lemasters J.J. Farber J.L. Jaeschke H. Toxicol. Appl. Pharmacol. 1990; 106: 165-178Google Scholar).At the cellular level, one of the earliest effects of oxidative stress is dissipation of transmembrane cation gradients (6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar) and sustained release of K+ ions (7Hallbrucker C. Ritter M. Lang F. Gerok W. Haussinger D. Eur. J. Biochem. 1993; 211: 449-458Google Scholar). In isolated livers, oxidative stress stimulates K+ efflux, increasing the concentration of K+ in perfusate from 5.6 to 13.1 mM (8Mets B. Hickman R. Allin R. van Dyk J. Lotz Z. Hepatology. 1993; 17: 668-676Google Scholar). Efflux of K+ appears early, preceding release of aminotransferases, and correlates closely with the fall in ATP levels (8Mets B. Hickman R. Allin R. van Dyk J. Lotz Z. Hepatology. 1993; 17: 668-676Google Scholar). Net loss of K+ ions is related in part to inhibition of Na+/K+ pump activity (4Rosser B.G. Gores G. Gastroenterology. 1995; 108: 252-275Google Scholar, 6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar). However, studies of other cell types have identified an additional and quantitatively important pathway for hypoxia-induced K+ efflux mediated by opening of K+ channels.In cardiovascular tissues, K+ channel opening represents an important adaptive response to stress since membrane hyperpolarization results in vasodilation and restoration of blood flow (9Silberberg S.D. van Breemen C. Pfluegers Arch. Eur. J. Physiol. 1992; 420: 118-120Google Scholar). Two modes of regulation have been identified, including opening of glibenclamide-sensitive KATP channels where ATP itself is responsible for channel gating (9Silberberg S.D. van Breemen C. Pfluegers Arch. Eur. J. Physiol. 1992; 420: 118-120Google Scholar, 10Komaru T. Lamping K.G. Eastham C.L. Dellsperger K.C. Circ. Res. 1991; 69: 1146-1151Google Scholar, 11Nichols C.G. Lederer W.J. Am. J. Physiol. 1991; 261: H1675-H1686Google Scholar) and opening of Ca2+-sensitive K+ channels in tissues where metabolic inhibition increases intracellular Ca2+ concentration ([Ca2+]i) (12Miller A.L. Morales E. LeBlanc N.R. Cole W.C. Am. J. Physiol. 1993; 265: H2184-H2195Google Scholar, 13Groschner K. Silberberg S.D. Gelband C.H. van Breemen C. Pfluegers Arch. Eur. J. Physiol. 1991; 417: 517-522Google Scholar). It is notable that metabolic inhibition releases Ca2+ from intracellular stores of liver cells (6Gasbarrini A. Borle A.B. Farghali H. Bender C. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 6654-6663Google Scholar, 14Lynch C.J. Deth R.C. Pharmacology. 1984; 28: 74-85Google Scholar, 15Tohkin M. Yoshimatsu N. Matsubara T. Jpn. J. Pharmacol. 1988; 46: 61-69Google Scholar), and the resulting increase in [Ca2+]i is characteristic of many forms of liver cell injury (4Rosser B.G. Gores G. Gastroenterology. 1995; 108: 252-275Google Scholar).The purpose of the current studies was to evaluate the relationship between cellular metabolism and membrane K+ permeability in HTC cells, a model liver cell line. Different models of metabolic inhibition increased K+ conductance 30-fold or more through activation of apamin-sensitive SKCa channels. Channel opening appears to occur through a mechanism distinct from cardiovascular tissues and involves translocation of protein kinase Cα (PKCα) from cytosol to membrane. Moreover, intracellular perfusion with purified PKCα activates currents in the absence of metabolic inhibition, suggesting that the α isoform of PKC may be selectively involved in channel opening.
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