FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2093354310> ?p ?o ?g. }

• W2093354310 endingPage "24717" @default.
• W2093354310 startingPage "24707" @default.
• W2093354310 abstract "The sphingolipid ceramide has been implicated in mediating cell death that is accompanied by mitochondrial functional alterations. Moreover, ceramide has been shown to accumulate in mitochondria upon induction of apoptotic processes. In this study, we sought to evaluate the effects of natural, highly hydrophobic long-chain ceramides on mitochondrial function in vitro. Ceramide in a dodecane/ethanol delivery system inhibited the opening of the mitochondrial permeability transition pore (PTP) induced by either oxidative stress, SH group cross-linking, or high Ca2+ load, suggesting that the inhibitory point is at a level at which major PTP regulatory pathways converge. Moreover, ceramide had no effect on well known mitochondrial components that modulate PTP activity, such as cyclophilin D, voltage-dependent anion channel, adenine nucleotide transporter, and ATP synthase. The inhibitory effect of ceramide on PTP was not stereospecific, nor was there a preference for ceramide over dihydroceramide. However, the effect of ceramide on PTP was significantly influenced by the fatty acid moiety chain length. These studies are the first to show that long-chain ceramide can influence PTP at physiologically relevant concentrations, suggesting that it is the only known potent natural inhibitor of PTP. These results suggest a novel mechanism of ceramide regulation of mitochondrial function. The sphingolipid ceramide has been implicated in mediating cell death that is accompanied by mitochondrial functional alterations. Moreover, ceramide has been shown to accumulate in mitochondria upon induction of apoptotic processes. In this study, we sought to evaluate the effects of natural, highly hydrophobic long-chain ceramides on mitochondrial function in vitro. Ceramide in a dodecane/ethanol delivery system inhibited the opening of the mitochondrial permeability transition pore (PTP) induced by either oxidative stress, SH group cross-linking, or high Ca2+ load, suggesting that the inhibitory point is at a level at which major PTP regulatory pathways converge. Moreover, ceramide had no effect on well known mitochondrial components that modulate PTP activity, such as cyclophilin D, voltage-dependent anion channel, adenine nucleotide transporter, and ATP synthase. The inhibitory effect of ceramide on PTP was not stereospecific, nor was there a preference for ceramide over dihydroceramide. However, the effect of ceramide on PTP was significantly influenced by the fatty acid moiety chain length. These studies are the first to show that long-chain ceramide can influence PTP at physiologically relevant concentrations, suggesting that it is the only known potent natural inhibitor of PTP. These results suggest a novel mechanism of ceramide regulation of mitochondrial function. In recent years, ceramide has become increasingly appreciated as a bioactive lipid that modulates apoptotic/necrotic cellular processes that are integrated at the mitochondrial level, as a result of studies demonstrating correlation between the intensity of cell death stimuli and the level of ceramide production (1Taha T.A. Mullen T.D. Obeid L.M. Biochim. Biophys. Acta. 2006; 1758: 2027-2036Crossref PubMed Scopus (224) Google Scholar, 2Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (621) Google Scholar, 3Mimeault M. FEBS Lett. 2002; 530: 9-16Crossref PubMed Scopus (67) Google Scholar, 4Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (705) Google Scholar). It has been suggested that ceramide conveys death signals to mitochondria by two mechanisms. One mechanism assumes selective permeabilization of the outer mitochondrial membrane for proapoptotic proteins as a process independent of inner membrane functions. The second mechanism presumes that loss of the permeability barrier of the inner mitochondrial membrane is a prerequisite for the subsequent outer membrane permeabilization. The simplest model of selective outer membrane permeabilization arises from experiments with artificial membranes (liposomes and black lipid membranes) (5Montes L.R. Ruiz-Arguello M.B. Goni F.M. Alonso A. J. Biol. Chem. 2002; 277: 11788-11794Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 6Siskind L.J. Colombini M. J. Biol. Chem. 2000; 275: 38640-38644Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 7Simon Jr., C.G. Gear A.R. Biochemistry. 1998; 37: 2059-2069Crossref PubMed Scopus (54) Google Scholar). Within this model, proapoptotic protein release due to large pore formation in the outer mitochondrial membrane is attributed to ceramide itself, whereas the inner membrane is viewed as being ceramide-insensitive. Although supported by extensive experimental material with isolated mitochondria (8Siskind L.J. Kolesnick R.N. Colombini M. J. Biol. Chem. 2002; 277: 26796-26803Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 9Siskind L.J. Kolesnick R.N. Colombini M. Mitochondrion. 2006; 6: 118-125Crossref PubMed Scopus (170) Google Scholar, 10Di Paola M. Zaccagnino P. Montedoro G. Cocco T. Lorusso M. J. Bioenerg. Biomembr. 2004; 36: 165-170Crossref PubMed Scopus (45) Google Scholar, 11Ghafourifar P. Klein S.D. Schucht O. Schenk U. Pruschy M. Rocha S. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), there are a number of studies in which ceramide alone did not affect cytochrome c release (12Kristal B.S. Brown A.M. J. Biol. Chem. 1999; 274: 23169-23175Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Yuan H. Williams C.D. Adachi S. Oltersdorf T. Gottlieb R.A. Mitochondrion. 2003; 2: 237-244Crossref PubMed Scopus (0) Google Scholar, 14Kashkar H. Wiegmann K. Yazdanpanah B. Haubert D. Kronke M. J. Biol. Chem. 2005; 280: 20804-20813Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Szalai G. Krishnamurthy R. Hajnoczky G. EMBO J. 1999; 18: 6349-6361Crossref PubMed Scopus (407) Google Scholar, 16Pastorino J.G. Tafani M. Rothman R.J. Marcinkeviciute A. Hoek J.B. Farber J.L. Marcineviciute A. J. Biol. Chem. 1999; 274: 31734-31739Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) or cytochrome c release was related to an inner membrane perturbation (15Szalai G. Krishnamurthy R. Hajnoczky G. EMBO J. 1999; 18: 6349-6361Crossref PubMed Scopus (407) Google Scholar, 16Pastorino J.G. Tafani M. Rothman R.J. Marcinkeviciute A. Hoek J.B. Farber J.L. Marcineviciute A. J. Biol. Chem. 1999; 274: 31734-31739Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 17Novgorodov S.A. Szulc Z.M. Luberto C. Jones J.A. Bielawski J. Bielawska A. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2005; 280: 16096-16105Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 18Arora A.S. Jones B.J. Patel T.C. Bronk S.F. Gores G.J. Hepatology. 1997; 25: 958-963Crossref PubMed Scopus (140) Google Scholar, 19Pastorino J.G. Simbula G. Yamamoto K. Glascott Jr., P.A. Rothman R.J. Farber J.L. J. Biol. Chem. 1996; 271: 29792-29798Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). This could be due to differences in cell types and experimental differences. Moreover, in Bax- and Bak-deficient cells, ceramide-induced cell death was completely suppressed (20Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1159) Google Scholar), indicating a likely role of Bcl-2 family proteins in ceramide-induced apoptosis. In fact it was recently shown that Bcl-2 can disassemble ceramide channels in the outer mitochondrial membrane (21Siskind L.J. Feinstein L. Yu T. Davis J.S. Jones D. Choi J. Zuckerman J.E. Tan W. Hill R.B. Hardwick J.M. Colombini M. J. Biol. Chem. 2008; 283: 6622-6630Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Another model of selective outer membrane permeabilization considers the Bcl-2 protein family localized in the cytoplasm and on the mitochondrial outer membrane to be major participants in the manifestation of the proapoptotic potential for ceramide. Activation of proapoptotic proteins, including Bid, Bax, and Bad, is mediated by interaction of ceramides with protein phosphatases PP1 and PP2A or with cathepsin D (22Chiang C.W. Kanies C. Kim K.W. Fang W.B. Parkhurst C. Xie M. Henry T. Yang E. Mol. Cell. Biol. 2003; 23: 6350-6362Crossref PubMed Scopus (125) Google Scholar, 23Ruvolo P.P. Deng X. Ito T. Carr B.K. May W.S. J. Biol. Chem. 1999; 274: 20296-20300Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 24Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4782) Google Scholar, 25Bidere N. Lorenzo H.K. Carmona S. Laforge M. Harper F. Dumont C. Senik A. J. Biol. Chem. 2003; 278: 31401-31411Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 26Xin M. Deng X. J. Biol. Chem. 2006; 281: 18859-18867Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 27Chiang C.W. Harris G. Ellig C. Masters S.C. Subramanian R. Shenolikar S. Wadzinski B.E. Yang E. Blood. 2001; 97: 1289-1297Crossref PubMed Scopus (129) Google Scholar, 28Heinrich M. Neumeyer J. Jakob M. Hallas C. Tchikov V. Winoto-Morbach S. Wickel M. Schneider-Brachert W. Trauzold A. Hethke A. Schutze S. Cell Death Differ. 2004; 11: 550-563Crossref PubMed Scopus (265) Google Scholar). However, the direct effect of ceramides on Bax binding/insertion has also been reported (14Kashkar H. Wiegmann K. Yazdanpanah B. Haubert D. Kronke M. J. Biol. Chem. 2005; 280: 20804-20813Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Birbes H. El Bawab S. Hannun Y.A. Obeid L.M. FASEB J. 2001; 15: 2669-2679Crossref PubMed Scopus (201) Google Scholar, 30Birbes H. Luberto C. Hsu Y.T. El Bawab S. Hannun Y.A. Obeid L.M. Biochem. J. 2005; 386: 445-451Crossref PubMed Scopus (110) Google Scholar). An increase in proapoptotic proteins bound to mitochondria is thought to trigger specific permeabilization of the outer mitochondrial membrane for cytochrome c and other mitochondrial intermembrane-resident apoptosis-inducing proteins (31Armstrong J.S. BioEssays. 2006; 28: 253-260Crossref PubMed Scopus (183) Google Scholar, 32Kroemer G. Galluzzi L. Brenner C. Physiol. Rev. 2007; 87: 99-163Crossref PubMed Scopus (2582) Google Scholar). A mechanism that accounts for participation of the inner mitochondrial membrane in the release of proapoptotic proteins involves the obligatory opening of the permeability transition pore in the inner membrane (PTP) 2The abbreviations used are:PTPpermeability transition poreΔΨmitochondrial inner membrane potentialCSAcyclosporine ACRCmitochondrial calcium retention capacityERendoplasmic reticulumMSmass spectrometryPhAsOphenylarsine oxidet-BuOOHt-butyl-hydroperoxideTPP+tetraphenylphosphonium. 2The abbreviations used are:PTPpermeability transition poreΔΨmitochondrial inner membrane potentialCSAcyclosporine ACRCmitochondrial calcium retention capacityERendoplasmic reticulumMSmass spectrometryPhAsOphenylarsine oxidet-BuOOHt-butyl-hydroperoxideTPP+tetraphenylphosphonium. (31Armstrong J.S. BioEssays. 2006; 28: 253-260Crossref PubMed Scopus (183) Google Scholar, 32Kroemer G. Galluzzi L. Brenner C. Physiol. Rev. 2007; 87: 99-163Crossref PubMed Scopus (2582) Google Scholar, 33Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2021) Google Scholar, 34Crompton M. J. Physiol. 2000; 529: 11-21Crossref PubMed Google Scholar). The resulting osmotic swelling of the mitochondrial inner membrane compartment leads to rupture of the outer mitochondrial membrane and release of proapoptotic proteins. It is thought that PTP opening occurs as a result of direct interaction of ceramide with the pore in the presence of Ca2+ (15Szalai G. Krishnamurthy R. Hajnoczky G. EMBO J. 1999; 18: 6349-6361Crossref PubMed Scopus (407) Google Scholar, 35Pacher P. Hajnoczky G. EMBO J. 2001; 20: 4107-4121Crossref PubMed Scopus (198) Google Scholar) or that PTP opening follows mitochondrial Ca2+ overload. In the latter scenario, the source of Ca2+ is the Ca2+ stores of the endoplasmic reticulum (ER), which are considered primary ceramide targets (20Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1159) Google Scholar, 36Pinton P. Ferrari D. Rapizzi E. Di Virgilio F. Pozzan T. Rizzuto R. EMBO J. 2001; 20: 2690-2701Crossref PubMed Scopus (469) Google Scholar). Support for this mechanism arises from experiments with many cell types in which ceramide-induced cell death is suppressed by PTP inhibitors, such as cyclosporine A (CSA) and bongkrekic acid (15Szalai G. Krishnamurthy R. Hajnoczky G. EMBO J. 1999; 18: 6349-6361Crossref PubMed Scopus (407) Google Scholar, 19Pastorino J.G. Simbula G. Yamamoto K. Glascott Jr., P.A. Rothman R.J. Farber J.L. J. Biol. Chem. 1996; 271: 29792-29798Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 35Pacher P. Hajnoczky G. EMBO J. 2001; 20: 4107-4121Crossref PubMed Scopus (198) Google Scholar, 37Stoica B.A. Movsesyan V.A. Lea P.M.T. Faden A.I. Mol. Cell Neurosci. 2003; 22: 365-382Crossref PubMed Scopus (125) Google Scholar, 38Lin C.F. Chen C.L. Chang W.T. Jan M.S. Hsu L.J. Wu R.H. Tang M.J. Chang W.C. Lin Y.S. J. Biol. Chem. 2004; 279: 40755-40761Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 39Gendron M.C. Schrantz N. Metivier D. Kroemer G. Maciorowska Z. Sureau F. Koester S. Petit P.X. Biochem. J. 2001; 353: 357-367Crossref PubMed Scopus (94) Google Scholar). permeability transition pore mitochondrial inner membrane potential cyclosporine A mitochondrial calcium retention capacity endoplasmic reticulum mass spectrometry phenylarsine oxide t-butyl-hydroperoxide tetraphenylphosphonium. permeability transition pore mitochondrial inner membrane potential cyclosporine A mitochondrial calcium retention capacity endoplasmic reticulum mass spectrometry phenylarsine oxide t-butyl-hydroperoxide tetraphenylphosphonium. Models of mitochondrial Ca2+ overload as a consequence of ceramide-induced Ca2+ release from the ER and the direct activation of PTP by ceramides were largely based on data obtained using artificial C2-ceramide. Whether these models are applicable in vivo with endogenously generated natural ceramide is unclear. Thus, traditional extrinsic and intrinsic pathways that involve endogenous ceramide generation (tumor necrosis factor, CD95/Fas/APO-1 (40Osawa Y. Uchinami H. Bielawski J. Schwabe R.F. Hannun Y.A. Brenner D.A. J. Biol. Chem. 2005; 280: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 41Paris F. Grassme H. Cremesti A. Zager J. Fong Y. Haimovitz-Friedman A. Fuks Z. Gulbins E. Kolesnick R. J. Biol. Chem. 2001; 276: 8297-8305Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 42Dbaibo G.S. El-Assaad W. Krikorian A. Liu B. Diab K. Idriss N.Z. El-Sabban M. Driscoll T.A. Perry D.K. Hannun Y.A. FEBS Lett. 2001; 503: 7-12Crossref PubMed Scopus (93) Google Scholar, 43Gamard C.J. Dbaibo G.S. Liu B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1997; 272: 16474-16481Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar); etoposide, staurosporine (44Sawada M. Nakashima S. Banno Y. Yamakawa H. Hayashi K. Takenaka K. Nishimura Y. Sakai N. Nozawa Y. Cell Death Differ. 2000; 7: 761-772Crossref PubMed Google Scholar, 45Ferraro C. Quemeneur L. Fournel S. Prigent A.F. Revillard J.P. Bonnefoy-Berard N. Cell Death Differ. 2000; 7: 197-206Crossref PubMed Google Scholar, 46Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Clin. Invest. 1999; 103: 971-978Crossref PubMed Google Scholar, 47Wiesner D.A. Dawson G. J. Neurochem. 1996; 66: 1418-1425Crossref PubMed Google Scholar)) are either Ca2+-insensitive (CD95/Fas/APO-1 (36Pinton P. Ferrari D. Rapizzi E. Di Virgilio F. Pozzan T. Rizzuto R. EMBO J. 2001; 20: 2690-2701Crossref PubMed Scopus (469) Google Scholar, 48Hampton M.B. Vanags D.M. Porn-Ares M.I. Orrenius S. FEBS Lett. 1996; 399: 277-282Crossref PubMed Scopus (95) Google Scholar), tumor necrosis factor (48Hampton M.B. Vanags D.M. Porn-Ares M.I. Orrenius S. FEBS Lett. 1996; 399: 277-282Crossref PubMed Scopus (95) Google Scholar, 49McFarlane S.M. Anderson H.M. Tucker S.J. Jupp O.J. MacEwan D.J. Mol. Cell Biochem. 2000; 211: 19-26Crossref PubMed Google Scholar)) or have limited sensitivity to Ca2+ (etoposide, staurosporine (20Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Crossref PubMed Scopus (1159) Google Scholar, 50Rabkin S.W. Kong J.Y. Cell Biol. Int. 2002; 26: 433-440Crossref PubMed Scopus (7) Google Scholar)), which might be related to interaction of Ca2+ with targets other than mitochondria (51Aoyama M. Grabowski D.R. Dubyak G.R. Constantinou A.I. Rybicki L.A. Bukowski R.M. Ganapathi M.K. Hickson I.D. Ganapathi R. Biochem. J. 1998; 336: 727-733Crossref PubMed Scopus (16) Google Scholar, 52Grabowski D.R. Dubyak G.R. Rybicki L. Hidaka H. Ganapathi R. Biochem. Pharmacol. 1998; 56: 345-349Crossref PubMed Scopus (12) Google Scholar). Thus, this model apparently more closely reflects “accidental” apoptosis during ischemia/reperfusion, which is accompanied by Ca2+ overload and oxidative stress and in which participation of PTP is expected (33Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2021) Google Scholar, 34Crompton M. J. Physiol. 2000; 529: 11-21Crossref PubMed Google Scholar). The critical role of PTP as a major apoptotic checkpoint has also been questioned. Recent studies have indicated that cells with inactivated PTP due to cyclophilin D (a main regulatory component of PTP and the CSA target) deficiency undergo normal apoptosis when challenged by variety of stimuli, including etoposide, staurosporine, tunicamycin, thapsigargin, brefeldin A, tumor necrosis factor, TRAIL, moderate Ca2+ overload, and Bax overexpression (53Schinzel A.C. Takeuchi O. Huang Z. Fisher J.K. Zhou Z. Rubens J. Hetz C. Danial N.N. Moskowitz M.A. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12005-12010Crossref PubMed Scopus (643) Google Scholar, 54Nakagawa T. Shimizu S. Watanabe T. Yamaguchi O. Otsu K. Yamagata H. Inohara H. Kubo T. Tsujimoto Y. Nature. 2005; 434: 652-658Crossref PubMed Scopus (1256) Google Scholar, 55Baines C.P. Kaiser R.A. Purcell N.H. Blair N.S. Osinska H. Hambleton M.A. Brunskill E.W. Sayen M.R. Gottlieb R.A. Dorn G.W. Robbins J. Molkentin J.D. Nature. 2005; 434: 658-662Crossref PubMed Scopus (1669) Google Scholar). Moreover, overexpression of cyclophilin D delays apoptosis induced by mild oxidative stress and staurosporine (56Lin D.T. Lechleiter J.D. J. Biol. Chem. 2002; 277: 31134-31141Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 57Li Y. Johnson N. Capano M. Edwards M. Crompton M. Biochem. J. 2004; 383: 101-109Crossref PubMed Scopus (126) Google Scholar). At the same time, cyclophilin D deficiency was protective under high intensity oxidative stress and high Ca2+ overload, conditions that precipitate necrosis. These data seem to rule out a critical role for PTP in both extrinsic and intrinsic apoptotic pathways but suggest that PTP opening is instrumental in necrosis. These studies raised questions about the extrapolation of results obtained with artificial short-chain ceramide analogs to in vivo studies and whether long- and short-chain ceramides affect the same binding sites on mitochondria with similar consequences. To address this question, we reinvestigated the effect of ceramides on the permeability of the inner mitochondrial membrane using natural long-chain ceramides delivered in dodecane/ethanol. This system had been successfully utilized for ceramide delivery to subcellular organelles in situ (41Paris F. Grassme H. Cremesti A. Zager J. Fong Y. Haimovitz-Friedman A. Fuks Z. Gulbins E. Kolesnick R. J. Biol. Chem. 2001; 276: 8297-8305Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 58Ardail D. Popa I. Bodennec J. Famy C. Louisot P. Portoukalian J. Biochim. Biophys. Acta. 2002; 1583: 305-310Crossref PubMed Scopus (11) Google Scholar) and to enzymes in vitro (59Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Our results show that long-chain but not short-chain ceramides suppress PTP activity at physiologically relevant concentrations. These studies raise the interesting possibility that long-chain ceramides promote apoptosis at the level of the mitochondria by causing PTP closure. Materials—Ceramides were from the Lipidomics Core of the Medical University of South Carolina; α-chymotrypsin (bovine pancreas) and cyclosporin A were from Calbiochem. All other reagents were from Sigma. Preparation of Mitochondria from Rat Liver—Liver mitochondria were isolated by differential centrifugation as described (60Graham J.M. Bonifacino J.S. Dasso M. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Current Protocols in Cell Biology. John Wiley & Sons, Inc., New York1999: 3.3.1-3.3.3Google Scholar, 61Frezza C. Cipolat S. Scorrano L. Nat. Protocols. 2007; 2: 287-295Crossref PubMed Scopus (717) Google Scholar) with some modifications. Briefly, mitochondria were prepared from livers of male Sprague-Dawley rats (220–250 g) fasted overnight. Livers from two rats were homogenized in 100 ml of isolation medium containing 230 mm mannitol, 70 mm sucrose, 2 mm EDTA, and 10 mm HEPES (pH 7.4 adjusted by KOH). Homogenate was centrifuged at 579 × gmax for 10 min to pellet the nucleus and unbroken cells. Supernatant from the previous step was centrifuged at 8,000 × gmax for 10 min to pellet mitochondria. The mitochondrial pellet was resuspended in isolation medium and centrifuged at 8,000 × gmax for 10 min. This step was repeated again, except that isolation medium contained 10 μm of EGTA instead of EDTA. The final mitochondrial pellet was resuspended in the above medium (final protein concentration 60 mg/ml). Where indicated, further mitochondrial purification was performed by self-generated Percoll gradient centrifugation (62Hoppel C.L. Kerner J. Turkaly P. Turkaly J. Tandler B. J. Biol. Chem. 1998; 273: 23495-23503Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Mitochondrial protein concentration was determined with a bicinchoninic acid assay using bovine serum albumin as a standard (63Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (17635) Google Scholar). Mitochondrial Incubation Medium—Unless otherwise specified, incubations of isolated mitochondria were conducted at 25 °C, using 1 mg/ml of protein in a medium containing 150 mm KCl, 10 mm HEPES (pH 7.4 adjusted by KOH), 5 mm succinate, 3 mm KH2PO4, and rotenone (2 μm). Deviations from this medium and other reagents employed are described in the figure legends. Mitochondrial membrane potential and swelling were followed simultaneously in a water-jacketed cell equipped with magnetic stirring. Ceramide Delivery—Ceramides were delivered in ethanol or in 2% dodecane solution in ethanol. In all experiments, ceramides were delivered as a 5-μl aliquot per 1 ml of incubation medium to maintain dodecane 0.01% (0.6 mm) and ethanol at 0.5% (0.1 m). The ceramide solution was warmed to 37 °C and introduced into the medium immediately after adding mitochondria. Mitochondrial Respiration—Oxygen consumption by mitochondria was measured in a chamber equipped with a Clark type oxygen electrode (Instech Laboratories) at the conditions described under “Mitochondrial Incubation Medium.” Measurement of Mitochondrial Permeabilization—Inner membrane permeabilization was assayed by measurements of ΔΨ and mitochondrial swelling. ΔΨ as estimated from the accumulation of TPP+ in the mitochondrial matrix as described by Kamo et al. (64Kamo N. Muratsuga M. Hongoh R. Kobatake Y. J. Membr. Biol. 1979; 49: 105-121Crossref PubMed Google Scholar). TPP+ (2 μm) was added to the incubation medium as indicated in the figure legends. Mitochondrial swelling was measured by changes in absorbance at 520 nm using a Brinkmann PC 900 probe colorimeter and a fiberoptic probe. Calcium Retention Capacity (CRC) of Mitochondria—CRC was evaluated according to previously published methods (65Soriano M.E. Nicolosi L. Bernardi P. J. Biol. Chem. 2004; 279: 36803-36808Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) in medium containing 250 mm sucrose, 10 mm Hepes-Tris, 5 mm succinate-Tris, 1 mm Pi-Tris, 10 μm EGTA-Tris, and 2 μm rotenone (pH 7.4) by gradual addition to mitochondria (1 mg/ml at 25 °C) of known amounts of Ca2+. The concentration of Ca2+ in the incubation medium was followed with a Ca2+-selective electrode (Orion). Cytochrome c Release from Mitochondria—Aliquots of mitochondrial suspension were taken as indicated in the figure legends and centrifuged at 15,000 × g for 3 min. The supernatants and mitochondria were frozen and stored at -80 °C. Cytochrome c in supernatants and mitochondria was quantified using the Quantikine cytochrome c ELISA kit (R & D Systems, Minneapolis, MN). Quantification was performed in duplicates according to the manufacturer's recommendations. Measurement of Mitochondrial Peptidyl-prolyl cis-trans Isomerase (Cyclophilin D) Activity—Mitochondria (200 μg/ml), untreated or treated with 5 μm C18-ceramide or 5 μm cyclosporin A at 25 °C for 15 min, were permeabilized for 5 min with alamethicin (7 μg/ml) and kept at 10 °C before the analysis. Peptidyl-prolyl cis-trans isomerase activity was assayed at 10 °C as described by Halestrap and Davidson (66Halestrap A.P. Davidson A.M. Biochem. J. 1990; 268: 153-160Crossref PubMed Google Scholar). Equal volumes of mitochondrial suspension and medium containing 0.52 mg/ml chymotrypsin were mixed, and the reading at A390 was allowed to stabilize for 2 min. The reaction was initiated by the addition of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (final concentration, 25 μm) from 25 mm stock dissolved in trifluoroethanol/LiCl (470 mm) (67Garcia-Echeverria C. Kofron J.L. Kuzmic P. Rich D.H. Biochem. Biophys. Res. Commun. 1993; 191: 70-75Crossref PubMed Scopus (26) Google Scholar). Mitochondrial Ceramide Measurements—Mitochondrial ceramide species were determined by tandem mass spectrometry (MS) (68Bielawski J. Szulc Z.M. Hannun Y.A. Bielawska A. Methods. 2006; 39: 82-91Crossref PubMed Scopus (396) Google Scholar). Mitochondria were incubated at 25 °C, using protein (1 mg/ml) in a medium containing 150 mm KCl, 10 mm HEPES (pH 7.4 adjusted by KOH), 5 mm succinate, 3 mm KH2PO4, and rotenone (2 μm). The incubations were performed in the absence or in the presence of C18-ceramide (1 nmol/mg protein). After 3 min, aliquots of mitochondrial suspension were taken and centrifuged at 15,000 × g for 3 min. Mitochondrial pellets were lysed in a buffer containing 10 mm Tris and 1% Triton X-100, pH 7.4, for electrospray ionization/MS/MS analysis of ceramides, which was performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer, operating in a multiple reaction-monitoring, positive ionization mode. Mitochondrial lysates, fortified with internal standards, were extracted with ethyl acetate/isopropyl alcohol/water (60:30:10, v/v/v), evaporated to dryness, and reconstituted in 100 μl of methanol. The samples were injected into the HP1100/TSQ 7000 liquid chromatography/MS system and gradient-eluted from the BDS Hypersil C8, 150 × 3.2-mm, 3-μm particle size column, with a 1.0 mm methanolic ammonium formate, 2 mm aqueous ammonium formate mobile phase system. The peaks for the target analytes and internal standards were collected and processed with the Xcalibur software system. Calibration curves were constructed by plotting peak area ratios of synthetic standards, representing each target analyte, to the corresponding internal standard. The target analyte peak area ratios from the samples were similarly normalized to their respective internal standard and compared with the calibration curves using a linear regression model. Statistical Analysis—The standard curve and the data for cytochrome c release were computed by generation of a four-parameter logistic curve fit. Where indicated, values were expressed as the mean value of the treatment groups ± S.D. Data were analyzed for statistically significant differences between groups using the two-tailed Student's t test. Statistical significance was ascribed to the data when p was <0.05. C18-ceramide Suppresses Ca2+-induced Permeability Transition and Increases Mitochondrial CRC—To determine whether natural long chain ceramide modulates the PTP, we used a dodecane/ethanol delivery system, which promotes the incorporation of long-chain ceramide into subcellular compartments (41Paris F. Grassme H. Cremesti A. Zager J. Fong Y. Haimovitz-Friedman A. Fuks Z. Gulbins E. Kolesnick R. J. Biol. Chem. 2001; 276: 8297-8305Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 58Ardail D. Popa I. Bodennec J. Famy C. Louisot P. Portoukalian J. Biochim. Biophys. Acta. 2002; 1583: 305-310Crossref PubMed Scopus (11) Google Scholar). Induction of permeability transition under conditions relevant to ischemia/reperfusion (presence of a large amount of Ca2+ and inorganic phosphate) was nearly completely suppressed by 1 nmol/mg protein C18-ceramide (Fig. 1). A 3-min preincubation with C18-ceramide resulted in 74% (736.4 ± 12.4 pmol/mg protein from 1 nmol added) incorporation into mitochondria as measured by MS. C18-ceramide prevented both the Ca2+-induced decline in ΔΨ (Fig. 1A, trace 2 versus trace 3) and large amplitude" @default.
• W2093354310 created "2016-06-24" @default.
• W2093354310 creator A5023831938 @default.
• W2093354310 creator A5036479060 @default.
• W2093354310 creator A5085128345 @default.
• W2093354310 date "2008-09-01" @default.
• W2093354310 modified "2023-10-11" @default.
• W2093354310 title "Long-chain Ceramide Is a Potent Inhibitor of the Mitochondrial Permeability Transition Pore" @default.
• W2093354310 cites W1480051253 @default.
• W2093354310 cites W1536767595 @default.
• W2093354310 cites W1554372630 @default.
• W2093354310 cites W1581414531 @default.
• W2093354310 cites W1583495578 @default.
• W2093354310 cites W1767614014 @default.
• W2093354310 cites W1964483580 @default.
• W2093354310 cites W1968112618 @default.
• W2093354310 cites W1970044546 @default.
• W2093354310 cites W1973472626 @default.
• W2093354310 cites W1976226379 @default.
• W2093354310 cites W1977884080 @default.
• W2093354310 cites W1981478938 @default.
• W2093354310 cites W1982687089 @default.
• W2093354310 cites W1983721382 @default.
• W2093354310 cites W1983755623 @default.
• W2093354310 cites W1984145308 @default.
• W2093354310 cites W1991429239 @default.
• W2093354310 cites W1993552956 @default.
• W2093354310 cites W1994442108 @default.
• W2093354310 cites W1997367774 @default.
• W2093354310 cites W1999952910 @default.
• W2093354310 cites W2000973050 @default.
• W2093354310 cites W2002487087 @default.
• W2093354310 cites W2002989164 @default.
• W2093354310 cites W2005571058 @default.
• W2093354310 cites W2006033770 @default.
• W2093354310 cites W2007688223 @default.
• W2093354310 cites W2008757391 @default.
• W2093354310 cites W2008888835 @default.
• W2093354310 cites W2009461852 @default.
• W2093354310 cites W2011297923 @default.
• W2093354310 cites W2011491573 @default.
• W2093354310 cites W2011587824 @default.
• W2093354310 cites W2011925577 @default.
• W2093354310 cites W2013765881 @default.
• W2093354310 cites W2014138744 @default.
• W2093354310 cites W2019524246 @default.
• W2093354310 cites W2021678802 @default.
• W2093354310 cites W2022791001 @default.
• W2093354310 cites W2022841230 @default.
• W2093354310 cites W2023555853 @default.
• W2093354310 cites W2024067398 @default.
• W2093354310 cites W2024149904 @default.
• W2093354310 cites W2027228594 @default.
• W2093354310 cites W2028900979 @default.
• W2093354310 cites W2029612038 @default.
• W2093354310 cites W2029641949 @default.
• W2093354310 cites W2029735625 @default.
• W2093354310 cites W2034554330 @default.
• W2093354310 cites W2040155866 @default.
• W2093354310 cites W2040802520 @default.
• W2093354310 cites W2044325325 @default.
• W2093354310 cites W2045122620 @default.
• W2093354310 cites W2045380458 @default.
• W2093354310 cites W2045454837 @default.
• W2093354310 cites W2048689532 @default.
• W2093354310 cites W2049187899 @default.
• W2093354310 cites W2050620574 @default.
• W2093354310 cites W2052406949 @default.
• W2093354310 cites W2053509579 @default.
• W2093354310 cites W2054149720 @default.
• W2093354310 cites W2054820743 @default.
• W2093354310 cites W2057194478 @default.
• W2093354310 cites W2057257618 @default.
• W2093354310 cites W2058811179 @default.
• W2093354310 cites W2059092928 @default.
• W2093354310 cites W2059246206 @default.
• W2093354310 cites W2063099262 @default.
• W2093354310 cites W2064364405 @default.
• W2093354310 cites W2065186260 @default.
• W2093354310 cites W2067033621 @default.
• W2093354310 cites W2068146558 @default.
• W2093354310 cites W2073229987 @default.
• W2093354310 cites W2073317184 @default.
• W2093354310 cites W2074800090 @default.
• W2093354310 cites W2074911197 @default.
• W2093354310 cites W2076269247 @default.
• W2093354310 cites W2076875353 @default.
• W2093354310 cites W2078702640 @default.
• W2093354310 cites W2078716275 @default.
• W2093354310 cites W2079828650 @default.
• W2093354310 cites W2088555531 @default.
• W2093354310 cites W2088629175 @default.
• W2093354310 cites W2090504233 @default.
• W2093354310 cites W2091387921 @default.
• W2093354310 cites W2091510369 @default.
• W2093354310 cites W2093361250 @default.
• W2093354310 cites W2096012158 @default.
• W2093354310 cites W2097767970 @default.

• next