Regional Loss of the Mitochondrial Membrane Potential in the Hepatocyte Is Rapidly Followed by Externalization of Phosphatidylserines at That Specific Site during Apoptosis

The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to ∼307 and also delayed the collapse of the MMP. This indicates that ATP was needed for the maintenance of the MMP probably via reverse action of the ATP synthase. In a subsequent study hepatocytes were incubated with Natural Killer cells for induction of apoptosis followed by addition of rotenone to deplete ATP. Under these conditions the PS staining co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. Moreover, exposure of PS was evenly distributed over the whole plasma membrane. In conclusion, we propose that after an apoptotic stimulus some mitochondria start to loose their MMP, which results in cessation of ATP production and perhaps even consumption of ATP. This results in an overall decrease in cellular ATP. ATP-consuming enzyme reactions most distal from still intact mitochondria will be most sensitive to such a decrease. Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme. The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to ∼307 and also delayed the collapse of the MMP. This indicates that ATP was needed for the maintenance of the MMP probably via reverse action of the ATP synthase. In a subsequent study hepatocytes were incubated with Natural Killer cells for induction of apoptosis followed by addition of rotenone to deplete ATP. Under these conditions the PS staining co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. Moreover, exposure of PS was evenly distributed over the whole plasma membrane. In conclusion, we propose that after an apoptotic stimulus some mitochondria start to loose their MMP, which results in cessation of ATP production and perhaps even consumption of ATP. This results in an overall decrease in cellular ATP. ATP-consuming enzyme reactions most distal from still intact mitochondria will be most sensitive to such a decrease. Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme. mitochondrial membrane potential interleukin-2-activated natural killer cells annexin V confocal laser scan microscopy propidium iodide phosphatidylserine(s) tetramethylrosamine 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-(3-methyl-2,3-dihydrobenzo-1,3-thiazolyl-2-methylidene)quinolinium] tetraiodide It has been demonstrated in many cell types, using a large variety of inducers, that mitochondria often play a crucial role in development of apoptosis and necrosis (reviewed in Refs. 1Kroemer G. Reed J.C. Nat. Med. 2000; 6: 509-513Google Scholar and 2Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Google Scholar). Already in the 1980s a relation was observed between elevated calcium concentrations and the mitochondrial membrane potential (MMP)1 on the one hand and cell death on the other hand upon exposure of hepatocytes to toxic compounds or after ischemia reperfusion (3Lemasters J.J. DiGiuseppi J. Nieminen A.L. Herman B. Nature. 1987; 325: 78-81Google Scholar). It was shown that opening of a high conductance permeability transition pore after oxidative stress in the mitochondrial inner membrane abruptly increases the permeability of the mitochondrial inner membrane to solutes of molecular mass up to 1500 Da (4Crompton M. Costi A. Hayat L. Biochem. J. 1987; 245: 915-918Google Scholar, 5Crompton M. Costi A. Eur. J. Biochem. 1988; 178: 489-501Google Scholar). The opening of the pore is associated with a collapse of the MMP, perturbation of intracellular and mitochondrial Ca2+ homeostasis, and subsequently cell death. The mitochondrial outer membrane is also the site of competition between the pro- and anti-apoptotic proteins of the Bcl-2 family and is associated with the opening of the pore (6Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsch T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Google Scholar, 7Bernardi P. Biochim. Biophys. Acta. 1998; 1366: 1-2Google Scholar, 8Zamzami N. Marchetti P. Castedo M. Hirsch T. Susin S.A. Masse B. Kroemer G. FEBS Lett. 1996; 384: 53-57Google Scholar, 9Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Google Scholar). Moreover, the mitochondria are a source of a number of the Bcl-2 family members and, in lymphoid cells, of the apoptosis inducing factor, a protein associated with opening of the pore and induction of apoptosis (10Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Google Scholar). Other mitochondrial factors that are associated with liver apoptosisin vivo are proteases that are released during cholestasis (11Gores G.J. Miyoshi H. Botla R. Aguilar H.I. Bronk S.F. Biochim. Biophys. Acta. 1998; 1366: 167-175Google Scholar). Finally several studies showed that after induction of apoptosis mitochondria release cytochrome c (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Google Scholar), which is associated with opening of the mitochondrial pore (13Petronelli V. Penzo D. Scorrano L. Bernardi P. Di Lisa F. J. Biol. Chem. 2001; 276: 12030-12034Google Scholar). Cytochrome cforms a complex with apoptotic protease activating factor-1, procaspase-9, and ATP. Formation of this complex, the apoptosome, leads to formation of active caspase-9, which that can subsequently activate other caspase proteins. In this way the apoptotic signal is amplified (reviewed in Ref. 14Bratton S.B. Cohen G.M. Trends Pharmacol Sci. 2001; 22: 306-315Google Scholar). Associated with the activation of the caspases is exposure of phosphatidyl- serines (PS) in the outer leaflet of cells (15Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 25-37Google Scholar, 16Blom W.M. De Bont H.J. Meijerman I. Kuppen P.J. Mulder G.J. Nagelkerke J.F. Hepatology. 1999; 29: 785-792Google Scholar). These molecules function as a signal for macrophages or other cells from the reticuloendothelial system to engulf and digest apoptotic bodies (17Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216Google Scholar). In this way release of intracellular components and a subsequent immunological reaction is prevented. The exposure of PS is either the result of inhibition of an ATP-dependent aminophospholipid transporter (18Williamson P. Schlegel R.A. Mol. Membr. Biol. 1994; 11: 199-216Google Scholar, 19Tang X. Schlegel R.A. Halleck M. Williamson P. Science. 1996; 272: 1495-1497Google Scholar) or activation of a calcium-dependent scramblase (20Williamson P. Kulick A. Zachowski A. Schlegel R.A. Devaux P.F. Biochemistry. 1992; 31: 6355-6360Google Scholar, 21Williamson P. Bevers E.M. Smeets E.F. Comfurius P. Schlegel R.A. Zwaal R.F.A. Biochemistry. 1995; 34: 10448-10455Google Scholar, 22Smeets E.F. Comfurius P. Bevers E.M. Zwaal R.F.A. Biochim. Biophys. Acta. 1994; 1195: 281-286Google Scholar). In addition, synthesis of PS through a calcium-dependent exchange of the polar head group of pre-existing phospholipids has been described (23Pelassy C. Breittmayer J.P. Aussel C. Biochim. Biophys. Acta. 2001; 1539: 256-264Google Scholar). In thymocytes exposure of PS occurs only in those cells that have lost their MMP (15Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 25-37Google Scholar, 24Kroemer G. Zamzani N. Susin S.A. Immunol. Today. 1997; 18: 44-51Google Scholar). In other cell types the opposite was found; in L929sAh cells transfected with the FAS receptor and treated with anti-FAS initially PS were exposed and followed later by a drop in MMP (25Denecker G. Dooms H. Van Loo G. Vercammen D. Grooten J. Fiers W. Declercq W. Vandenabeele P. FEBS Lett. 2000; 465: 47-52Google Scholar). We described the involvement of the mitochondria in the induction of apoptosis in hepatocytes after attack by interleukin-activated Natural Killer (A-NK) cells (16Blom W.M. De Bont H.J. Meijerman I. Kuppen P.J. Mulder G.J. Nagelkerke J.F. Hepatology. 1999; 29: 785-792Google Scholar). Fluorescence-activated cell sorter analysis after staining for extracellular-oriented PS with fluorescent-labeled annexin V (ANV) demonstrated that A-NK cells induced apoptosis in the hepatocytes (107 apoptotic cells after 30 min and 387 after 60 min of co-incubation). In these apoptotic cells the overall MMP was ∼607 of the value as compared with non-apoptotic hepatocytes. The decrease in MMP and exposure of PS in hepatocytes is apparently tightly coupled, because all cells with a lowered MMP exposed PS. In comparison with other studies (15Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 25-37Google Scholar, 24Kroemer G. Zamzani N. Susin S.A. Immunol. Today. 1997; 18: 44-51Google Scholar, 25Denecker G. Dooms H. Van Loo G. Vercammen D. Grooten J. Fiers W. Declercq W. Vandenabeele P. FEBS Lett. 2000; 465: 47-52Google Scholar) the decrease in MMP was relatively moderate, and the exposure of PS was very rapid. Microscopical examination showed that ANV staining was unevenly distributed over the cell membrane. To gain more insight in the spatio-temporal relationship between a decrease in the MMP on the one hand and exposure of PS on the other hand the present study was undertaken. Time-lapse video microscopy and confocal laser scan microscopy of hepatocytes that were attacked by A-NK cells was performed. In addition, two totally unrelated apoptosis-inducing stimuli were applied: addition of extracellular ATP or cycloheximide (26Nagelkerke J.F. Zoetewey J.P. Lemasters J.J. Nieminnen A.L. Mitochondria in Pathogenesis. Kluwer Academic/Plenum Publishing Corp., New York2001: 449-464Google Scholar, 27Blom W.M. De Bont H.J.G.M. Meijerman I. Mulder G.J. Nagelkerke J.F. Biochem. Pharmacol. 1999; 58: 1891-1898Google Scholar). We report that in all cases a local loss of MMP of a limited number of mitochondria in the cell results, within minutes, in exposure of phosphatidylserines at that particular spot. Collagenase, recombinant protein ANV, and HEPES were obtained from Roche Molecular Biochemicals. Tetramethylrosamine (TMR), rhodamine 123, propidium iodide (PI), TOTO-3, and the AlexaTM protein labeling kit were from Molecular Probes. Fluorescent ANV was prepared by labeling with AlexaTM488 or AlexaTM633. Bovine the and were from complex was prepared as described J.F. Van J. Biol. Chem. Scholar). of natural killer cells and activation of these cells were as described (16Blom W.M. De Bont H.J. Meijerman I. Kuppen P.J. Mulder G.J. Nagelkerke J.F. Hepatology. 1999; 29: 785-792Google Scholar). a cell was prepared from isolated from a a from The were from other cells cells, by The cells ∼307 A-NK cells, which were and in with and The was with recombinant and cells were and the cells, A-NK cells, were for The obtained for of cells and for of cell cells were isolated by from A. Nagelkerke J.F. Van Van J. G.J. Kuppen 1997; Scholar), from and at at the of the The were and at a to the the were for of the freshly isolated cells was as by cells were on To and the by A-NK cells, the complex protein of hepatocytes was with the W.M. De Bont H.J.G.M. Meijerman I. Mulder G.J. Nagelkerke J.F. Biochem. Pharmacol. 1999; 58: 1891-1898Google Scholar). This leads to of the cell as and activation of the A. Nagelkerke J.F. Van Van J. G.J. Kuppen 1997; Scholar, B. J. Immunol. 1998; Scholar). The liver cells were with the at for min in of HEPES with and and for 30 min with cells were and in either with or the with isolated liver cells were to to with in (16Blom W.M. De Bont H.J. Meijerman I. Kuppen P.J. Mulder G.J. Nagelkerke J.F. Hepatology. 1999; 29: 785-792Google Scholar). The were in a and of cell was to the the hepatocytes were to for The cells were at the The with the A-NK cells were by of the and addition of A-NK cells in with in the of This was because showed that resulted in apoptosis in a large number of hepatocytes (16Blom W.M. De Bont H.J. Meijerman I. Kuppen P.J. Mulder G.J. Nagelkerke J.F. Hepatology. 1999; 29: 785-792Google Scholar). In other ATP or cycloheximide were to the were in the on a that was at were incubated at a of the were for and ATP The video microscopy system of an with a and a ANV staining was using a for a and a was using a for a and a for were using a by a confocal laser an system was J.F. De Bont H.J.G.M. J. 1996; Scholar). The of the a and an ANV was with the with the and with the of PS on the extracellular of the plasma membrane of hepatocytes was by staining PS with ANV with AlexaTM488 ANV and AlexaTM488 in a complex of (4Crompton M. Costi A. Hayat L. Biochem. J. 1987; 245: 915-918Google Scholar). ANV and of a of the were to of cell in the of the were for min with ANV and were of the of the apoptotic stimuli were of the was by the in and of a using These were using The result of this analysis is a number between and The indicates and the indicates J. A. D. Scholar). of intracellular calcium the hepatocytes were with for 30 the cells were with at ANV and A-NK cells were a group of cells the after and were using a of and a were by of the of the were by of the by the on a The intracellular calcium was using the with as the for Ca2+ and is the at is the at is the is the The MMP of and cells was by analysis using ANV, and rhodamine as described (16Blom W.M. De Bont H.J. Meijerman I. Kuppen P.J. Mulder G.J. Nagelkerke J.F. Hepatology. 1999; 29: 785-792Google Scholar). after addition of ANV, rhodamine 123, and the hepatocytes were incubated for min on in the The of cells was using a using the of the hepatocytes were in of ATP was by addition of to the and after min was with The were and ATP was in the using the are as The was with an isolated hepatocytes were with to on the in addition, ANV with was to PS. The hepatocytes were to a at the of the after of the A-NK cells were as a to the and some a between the hepatocytes and A-NK cells been that min after the addition of the A-NK cells such a was this the of the was still min later the apoptotic this at the MMP was This was followed min later by the staining of PS These decrease in MMP and externalization of was the of a staining in the indicates that min necrosis the cells were still The of the signal and the PS signal are shown in the these the signal The of staining indicates that only at the where mitochondria lost their MMP externalization of PS To more we the were incubated the conditions as described the MMP, and the externalized PS. can be in the the not the that PS only in the of The was using which is at the of the To the of the apoptosis was induced in hepatocytes with a protein synthesis a min after the addition of cycloheximide. The in at the apoptotic These cells have a lowered MMP and are high in ANV the indicates of the two results were apoptosis was induced using addition of extracellular ATP These results that three different apoptotic the externalization of PS in the of for this be a local decrease in ATP The enzyme that the of aminophospholipid ATP for The most to the role of ATP have been intracellular of the using a those for of Ca2+ or MMP. such a does not we of ATP collapse of the MMP by using the mitochondrial inhibitor in of the of rotenone on intracellular ATP and MMP was were and at different two were was for ATP The was incubated with ANV, rhodamine 123, and for analysis as described and that even at rotenone ATP were of within 30 In the MMP remained intact for more addition of fructose prevented the decrease in ATP to and delayed the decrease in the MMP. in the number of cells as a result of rotenone exposure in with the decrease in MMP. all in the in the cells incubated with rotenone more of the cells were indicating that a decrease in cellular ATP is not to hepatocytes were with A-NK cells to and after min rotenone was to deplete ATP, and the of PS staining and MMP staining was in apoptotic the MMP, and the of the PS. In were is that To this the of this and of the in were The value in was whereas the value for was This indicates that was much more in staining after with comparison of and that PS staining was in the whereas in cells the whole plasma membrane was hepatocytes were incubated with rotenone and fructose and A-NK cells were obtained in which MMP and PS staining were The of the decrease in ATP was not to the PS In the of rotenone and fructose or rotenone the of the cells apoptotic and of is on laser scan of of the MMP and the externalization of PS in isolated hepatocytes exposed to cycloheximide. were the as for were incubated with 30 is apoptotic were exposed to cycloheximide for concentrations in hepatocytes and to fructose were and were of the was in In this ATP was using the In the other of the the MMP was using the rotenone and laser scan of of the MMP and the externalization of PS in isolated hepatocytes exposed to A-NK cells after addition of were the as for was with laser and ANV was with laser These were and The is shown in apoptotic were exposed to A-NK cells for was min after the start of the To the role of intracellular calcium hepatocytes were with and incubated with the A-NK cells in the of the was as in video in many cells an in the intracellular calcium was but in the signal at the cell the calcium were within the a was to to more We that addition of A-NK resulted in a of from the of to in of of the observed hepatocytes followed by externalization of the PS. in the other in intracellular calcium PS were Moreover, in of the hepatocytes PS were not externalized a Apoptosis was induced in the hepatocytes after attack by A-NK cells (16Blom W.M. De Bont H.J. Meijerman I. Kuppen P.J. Mulder G.J. Nagelkerke J.F. Hepatology. 1999; 29: 785-792Google Scholar) and exposure to ATP (26Nagelkerke J.F. Zoetewey J.P. Lemasters J.J. Nieminnen A.L. Mitochondria in Pathogenesis. Kluwer Academic/Plenum Publishing Corp., New York2001: 449-464Google Scholar) or cycloheximide W.M. De Bont H.J.G.M. Meijerman I. Mulder G.J. Nagelkerke J.F. Biochem. Pharmacol. 1999; 58: 1891-1898Google Scholar). three conditions resulted in a rapid decrease of the MMP followed by externalization of the PS. shown in the of the attack by A-NK cells, between some hepatocytes and A-NK cells was after Already at min mitochondria in some areas of the hepatocytes lost their MMP, and at min exposure of PS at with mitochondria is from the externalization of PS at those areas or of the cell where mitochondria lost their MMP. The most for PS externalization at these is that ATP in the of mitochondria in inhibition of the translocase that phosphatidylserines to the inner are to local in ATP within the ATP has been in but this of and of the cells in which are conditions that will probably membrane A. Biochem. J. Scholar) and of hepatocytes with A-NK of a for P. A. M. R. Biol. 2001; Scholar) will membrane and, in addition, cell that in hepatocytes. to discriminate between of a collapse of the MMP itself or the decrease in ATP, we the to deplete ATP from cells the MMP by of the cells with we ATP with rotenone in the of fructose to Under conditions the staining for externalized PS co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. We as P. M. E. Lett. 1998; Scholar), that of ATP by itself does not an apoptotic signal is B. V. S. B. FEBS Lett. Scholar) a was PS exposure was by of intracellular ATP. In cells incubated with rotenone in the of fructose the ATP remained the MMP was at the as in cells indicating that ATP from fructose is for maintenance of the MMP. a has been described in cells with mitochondria and is on reverse action of the ATP S. J. D.P. J. Biol. Chem. 1999; Scholar). The enzyme that ATP from is the and often in the of mitochondria J. Sci. Scholar). these have to ATP. We that ATP is for maintenance of the MMP to of the a of the mitochondrial in a cell lost potential in in a of cells that have a high overall MMP but PS This is in with the in thymocytes and cells (6Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsch T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Google Scholar, M. Hirsch T. Susin S.A. Zamzami N. Marchetti P. Macho A. Kroemer G. J. Immunol. 1996; Scholar) in which the MMP to be externalization of PS we that the between MMP and PS is a local drop in ATP. The of cells to ATP by mitochondria or and the of ATP consumption the local ATP In local consumption of ATP by mitochondria and the of high ATP consumption by the plasma membrane and at the result in a in this in the of the cell M. J. Scholar). This between different cell types and, the coupling between MMP and PS. the translocase is also an ATP-dependent enzyme this where the mitochondria not function PS externalization to ATP, calcium-dependent have also been described to play a role in PS externalization by activation of a calcium-dependent scramblase or synthesis of PS through a calcium-dependent exchange of the polar head group of pre-existing We the calcium in hepatocytes with during attack by A-NK ANV and were to the and, we cells apoptotic or We relation between of intracellular calcium and PS exposure. In of the hepatocytes calcium was elevated exposure of but in was in a number of cells, the calcium to PS exposure In addition, some hepatocytes elevated calcium but not PS. in the with the A-NK cells the activation of a scramblase is probably not be the involvement of calcium-dependent synthesis is In we that in hepatocytes during apoptosis mitochondria in areas within the cell their MMP in local exposure of the PS whereas in other areas mitochondria the apparently leads to exposure of which is for of the apoptotic the be for of cytochrome apoptotic protease activating factor-1, procaspase-9, and ATP an We in this study A-NK cells that the apoptosis-inducing molecules and FAS and necrosis apoptosis-inducing In addition, ATP was which an of in a loss of MMP J.P. B. Bont H.J.G.M. Mulder G.J. Nagelkerke J.F. Biochem. J. 1992; Scholar) and cycloheximide that is a protein synthesis these stimuli the and, the spatio-temporal relationship between a decrease in MMP and exposure of PS be a in cells that on mitochondria for their ATP