Impaired Regulation of Brain Mitochondria by Extramitochondrial Ca2+ in Transgenic Huntington Disease Rats
2008; Elsevier BV; Volume: 283; Issue: 45 Linguagem: Inglês
10.1074/jbc.m709555200
ISSN1083-351X
AutoresFrank N. Gellerich, Zemfira Gizatullina, Huu Phuc Nguyen, Sonata Trumbeckaitė, Stefan Vielhaber, Enn Seppet, Stephan Zierz, G. Bernhard Landwehrmeyer, Olaf Rieß, Stephan von Hörsten, Frank Striggow,
Tópico(s)Neurological disorders and treatments
ResumoHuntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt51Q), modeling the adult form of HD. (Cafree2+) up to 2 μm activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. (Cafree2+) above 2 μm inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, did not affect the Ca2+-dependent activation of respiration but reduced Ca2+-induced inhibition. Thus, Ca2+ activation was mediated exclusively by extramitochondrial Ca2+, whereas inhibition was promoted also by intramitochondrial Ca2+. In contrast, htt51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+ activation, and a higher susceptibility to Ca2+-dependent inhibition. Furthermore htt51Q mitochondria exhibited a diminished membrane potential stability in response to Ca2+, lower capacities and rates of Ca2+ accumulation, and a decreased Ca2+ threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca2+-induced inhibition of respiration of htt51Q mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca2+. In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca2+. We suggest that specific regulatory Ca2+ binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt51Q and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD. Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt51Q), modeling the adult form of HD. (Cafree2+) up to 2 μm activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. (Cafree2+) above 2 μm inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, did not affect the Ca2+-dependent activation of respiration but reduced Ca2+-induced inhibition. Thus, Ca2+ activation was mediated exclusively by extramitochondrial Ca2+, whereas inhibition was promoted also by intramitochondrial Ca2+. In contrast, htt51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+ activation, and a higher susceptibility to Ca2+-dependent inhibition. Furthermore htt51Q mitochondria exhibited a diminished membrane potential stability in response to Ca2+, lower capacities and rates of Ca2+ accumulation, and a decreased Ca2+ threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca2+-induced inhibition of respiration of htt51Q mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca2+. In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca2+. We suggest that specific regulatory Ca2+ binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt51Q and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD. Huntington disease (HD) 2The abbreviations used are: HD, Huntington disease; BSA, bovine serum albumin; CsA, cyclosporin A; [Ca2+]cyt, cytosolic Ca2+; htt, huntingtin; httexpQ, huntingtin with expanded poly(Q) tract; httnQ, huntingtin with n glutamine repeats; PT, permeability transition; RR, ruthenium red; WT, wild type; ΔΨ, mitochondrial membrane potential; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; IM, isolation medium; MOPS, 4-morpholinepropanesulfonic acid; suc, succinate; mal, malate; glu, glutamate; pyr, pyruvate; AOA, aminooxyacetate; RCI, respiratory control index. 2The abbreviations used are: HD, Huntington disease; BSA, bovine serum albumin; CsA, cyclosporin A; [Ca2+]cyt, cytosolic Ca2+; htt, huntingtin; httexpQ, huntingtin with expanded poly(Q) tract; httnQ, huntingtin with n glutamine repeats; PT, permeability transition; RR, ruthenium red; WT, wild type; ΔΨ, mitochondrial membrane potential; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; IM, isolation medium; MOPS, 4-morpholinepropanesulfonic acid; suc, succinate; mal, malate; glu, glutamate; pyr, pyruvate; AOA, aminooxyacetate; RCI, respiratory control index. is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the coding region of the huntingtin (htt) gene resulting in an expanded polyglutamine stretch in the htt protein (httexpQ) (1Ambrose C.M. Duyao M.P. Barnes G. Bates G.P. Lin C.S. Srinidhi J. Baxendale S. Hummerich H. Lehrach H. Altherr M. Somat. Cell Mol. Genet. 1994; 20: 27-38Crossref PubMed Scopus (227) Google Scholar, 2Li S.H.L. Schilling G. Young III, W.S. Li X.J. Margolis R.L. Stine O.C. Wagster M.V. Abbott M.H. Franz M.L. Ranen N.G. Folstein S.E. Hedreen J.C. Ross C.A. Neuron. 1993; 11: 985-993Abstract Full Text PDF PubMed Scopus (274) Google Scholar). The CAG repeat length of httexpQ correlates inversely with the time point of disease onset (3Langbehn D.R. Brinkman R.R. Falush D. Paulsen J.S. Hayden M.R. Clin. Genet. 2004; 65: 276-277Crossref Scopus (591) Google Scholar). Unmodified htt and even httexpQ in HD are abundantly expressed in most tissues (2Li S.H.L. Schilling G. Young III, W.S. Li X.J. Margolis R.L. Stine O.C. Wagster M.V. Abbott M.H. Franz M.L. Ranen N.G. Folstein S.E. Hedreen J.C. Ross C.A. 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Kazantsev A. Marsh J.L. Sullivan P.G. Steffan J.S. Sensi S.L. Thompson L.M. Hum. Mol. Genet. 2007; 16: 61-77Crossref PubMed Scopus (213) Google Scholar). Indeed decreased Ca2+ accumulation capacities of mitochondria from brain of YAC72Q mice (7Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Nat. Neurosci. 2002; 5: 731-736Crossref PubMed Scopus (854) Google Scholar), from skeletal muscle of htt140Q R6/2 mice (8Gizatullina Z.Z. Lindenberg K.S. Harjes P. Chen Y. Kosinski C.M. Landwehrmeyer B.G. Ludolph A.C. Striggow F. Zierz S. Gellerich F.N. Ann. Neurol. 2006; 59: 407-411Crossref PubMed Scopus (66) Google Scholar), from liver of htt111Q mice (9Choo Y.S. Johnson G.V. MacDonald M. Detloff P.J. Lesort M. Hum. Mol. Genet. 2004; 13: 1407-1420Crossref PubMed Scopus (406) Google Scholar), from HD patient's lymphocytes (7Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Nat. 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Nicholls D.G. J. Neurochem. 2007; 101: 241-249Crossref PubMed Scopus (114) Google Scholar). Recently we have presented the first experimental evidence for an impaired oxidative phosphorylation in isolated HD mitochondria from skeletal muscle of R6/2 mice after their exposure to elevated Ca2+ levels (8Gizatullina Z.Z. Lindenberg K.S. Harjes P. Chen Y. Kosinski C.M. Landwehrmeyer B.G. Ludolph A.C. Striggow F. Zierz S. Gellerich F.N. Ann. Neurol. 2006; 59: 407-411Crossref PubMed Scopus (66) Google Scholar). Similarly in situ measurements of mitochondrial respiration revealed a declined oxidative phosphorylation in HD150Q striatal cells but only after N-methyl-d-aspartate receptor-induced Ca2+ stress (13Oliveira J.M. Jekabsons M.B. Chen S. Lin A. Rego A.C. Goncalves J. Ellerby L.M. Nicholls D.G. J. Neurochem. 2007; 101: 241-249Crossref PubMed Scopus (114) Google Scholar). 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Biochemistry. 1997; 36: 7071-7080Crossref PubMed Scopus (88) Google Scholar) activate mitochondrial respiration due to stimulation of mitochondrial dehydrogenases (16Hansford R.G. Zorov D. Mol. Cell. Biochem. 1998; 184: 359-369Crossref PubMed Google Scholar). Elevated intramitochondrial Ca2+ levels also induce permeability transition (PT) if distinct [Ca2+]cyt thresholds are exceeded (17Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar). In addition, a reduced mitochondrial membrane potential (ΔΨ) and a decreased redox pressure, defined by the ratio of NADH/NAD+, further reinforce the tendency for undergoing mitochondrial PT (17Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar). Recently activation of the mitochondrial malate/aspartate shuttle was observed at low, i.e. submicromolar, [Ca2+]cyt. This effect was caused by a Ca2+-dependent stimulation of the glutamate/aspartate translocator (18Pardo B. Contreras L. Serrano A. Ramos M. Kobayashi K. Iijima M. Saheki T. Satrustegui J. J. Biol. Chem. 2006; 281: 1039-1047Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Palmieri L. Pardo B. Lasorsa F.M. del Arco A. Kobayashi K. Iijima M. Runswick M.J. Walker J.E. Saheki T. Satrustegui J. Palmieri F. EMBO J. 2001; 20: 5060-5069Crossref PubMed Scopus (392) Google Scholar). This carrier, termed aralar in brain mitochondria, was shown to also be involved in the transport of reducing hydrogen into mitochondria via a reconstituted malate/aspartate shuttle (18Pardo B. Contreras L. Serrano A. Ramos M. Kobayashi K. Iijima M. Saheki T. Satrustegui J. J. Biol. Chem. 2006; 281: 1039-1047Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Considering this background, it was the first goal of this study to answer the question whether or not httexpQ is mitochondriotoxic and if so to identify the underlying pathomechanism. For this purpose, we investigated the mitochondrial function in a newly generated transgenic HD rat strain with 51 glutamine repeats (htt51Q) (20von Hörsten S. Schmitt I. Nguyen H.P. Holzmann C. Schmidt T. Walther T. Bader M. Pabst R. Kobbe P. Krotova J. Stiller D. Kask A. Vaarmann, A, Rathke-Hartlieb S. Schulz J.B. Grasshoff U. Bauer I. Vieira-Saecker A.M. Paul M. Jones L. Lindenberg K.S. Landwehrmeyer B. Bauer A. Li X.J. Riess O. Hum. Mol. Genet. 2003; 12: 617-624Crossref PubMed Scopus (292) Google Scholar). In contrast to httQ150 R6/2 mice, a model of the juvenile form of HD (21Mangiarini L. Sathasivam K. Seller M. Cozens B. Harper A. Hetherington C. Lawton M. Trottier Y. Lehrach H. Davies S.W. Bates G.P. Cell. 1996; 87: 493-506Abstract Full Text Full Text PDF PubMed Scopus (2583) Google Scholar), the htt51Q rat strain exhibits specifically an adult-related onset of the neurological HD phenotype (20von Hörsten S. Schmitt I. Nguyen H.P. Holzmann C. Schmidt T. Walther T. Bader M. Pabst R. Kobbe P. Krotova J. Stiller D. Kask A. Vaarmann, A, Rathke-Hartlieb S. Schulz J.B. Grasshoff U. Bauer I. Vieira-Saecker A.M. Paul M. Jones L. Lindenberg K.S. Landwehrmeyer B. Bauer A. Li X.J. Riess O. Hum. Mol. Genet. 2003; 12: 617-624Crossref PubMed Scopus (292) Google Scholar). Because HD-specific changes in mitochondrial function may be related to alterations in the intracellular Ca2+ homeostasis (7Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Nat. Neurosci. 2002; 5: 731-736Crossref PubMed Scopus (854) Google Scholar, 8Gizatullina Z.Z. Lindenberg K.S. Harjes P. Chen Y. Kosinski C.M. Landwehrmeyer B.G. Ludolph A.C. Striggow F. Zierz S. Gellerich F.N. Ann. Neurol. 2006; 59: 407-411Crossref PubMed Scopus (66) Google Scholar, 9Choo Y.S. Johnson G.V. MacDonald M. Detloff P.J. Lesort M. Hum. Mol. Genet. 2004; 13: 1407-1420Crossref PubMed Scopus (406) Google Scholar, 10Milakovic T. Quintanilla R.A. Johnson G.V. J. Biol. Chem. 2006; 281: 34785-34795Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11Rockabrand E. Slepko N. Pantalone A. Nukala V.N. Kazantsev A. Marsh J.L. Sullivan P.G. Steffan J.S. Sensi S.L. Thompson L.M. Hum. Mol. Genet. 2007; 16: 61-77Crossref PubMed Scopus (213) Google Scholar, 22Bezprozvanny I. Hayden M.R. Biochem. Biophys. Res. Commun. 2004; 322: 1310-1317Crossref PubMed Scopus (208) Google Scholar, 23Tang T.S. Slow E. Lupu V. Stavrovscaya I.G. Sugimori M. Llinas R. Kristal B.S. Hayden M.R. Bezprozvanny I. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2602-2607Crossref PubMed Scopus (311) Google Scholar), the second goal of this study was to evaluate the influence of extramitochondrial Ca2+ on the function of wild type (WT) and htt51Q mitochondria. We studied the effects of extramitochondrial Ca2+ on mitochondrial Ca2+ accumulation and oxidative phosphorylation in mitochondria isolated from striatum and brain. We found that extramitochondrial Ca2+ in the submicromolar range was able to activate mitochondrial glutamate uptake, probably via binding on the high affinity Ca2+ binding sites of the aspartate/glutamate translocator (aralar) that are exposed into the mitochondrial intermembrane space. Htt51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+ activation, and a higher susceptibility to Ca2+-dependent inhibition. Furthermore htt51Q mitochondria exhibited a diminished membrane potential stability in response to Ca2+, lower capacities and rates of Ca2+ accumulation, and a decreased Ca2+ threshold for PT in a substrate-independent but CsA-sensitive manner. Compared with WT, Ca2+-induced inhibition of respiration of htt51Q mitochondria was less sensitive to ruthenium red (RR), indicating the involvement of extramitochondrial Ca2+. In summary, our data suggest that mitochondriotoxic actions of htt51Q might be realized by affecting the regulatory Ca2+ binding sites of mitochondrial carrier proteins like aralar and the PT pore, finally leading to energetic depression (6Seppet E. Gizatullina Z. Trumbeckaite S. Zierz S. Striggow F. Gellerich F.N. Saks V. Molecular System Bioenergetics, Energy for Life. Wiley-VCH, Weinheim, Germany2007: 479-520Crossref Scopus (9) Google Scholar, 24Gellerich F.N. Trumbeckaite S. Müller T. Chen Y. Deschauer M. Gizatullina Z. Zierz S. Mol. Cell. Biochem. 2004; 256/257: 391-405Crossref Google Scholar), mitochondrial cell death, and atrophy of affected tissues (6Seppet E. Gizatullina Z. Trumbeckaite S. Zierz S. Striggow F. Gellerich F.N. Saks V. Molecular System Bioenergetics, Energy for Life. Wiley-VCH, Weinheim, Germany2007: 479-520Crossref Scopus (9) Google Scholar). Animals—Transgenic animals were obtained from the central animal facility of the University of Tübingen, Tübingen, Germany. HD rats expressed 727 amino acids of the htt51Q gene corresponding to 22% of the full-length gene. Tail tips were removed from all rats at the age of 3 weeks, and genotypes were determined by Southern blot analysis. We used male and female rats aged 21–27 months and compared them with age-matched littermate WT rats from the same source. For some studies, normal adult Wistar WU rats were also used (Charles River Laboratories). All research and animal care procedures were performed according to European guidelines. Isolation of Mitochondria—Mitochondria were prepared either from striatum or from the remaining part of the brain (total brain minus striatum) using a slightly modified protocol according to Kudin et al. (25Kudin A.P. Bimpong-Buta N.Y. Vielhaber S. Elger C.E. Kunz W.S. J. Biol. Chem. 2004; 279: 4127-4135Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar), avoiding bovine serum albumin (BSA) during the preparation and investigation of mitochondria. In brief, animals were anesthetized with CO2 and killed by decapitation. Brain tissue was immediately transferred into the ice-cold isolation medium (IM) consisting of 225 mm mannitol, 75 mm sucrose, 20 mm MOPS, 1 mm EGTA, and 0.5 mm dithiothreitol, pH 7.4. Then we minced the tissue, added 10 ml of nargase medium (IM + 0.05% (w/v) nargase)/1 g of tissue, and homogenized the mixture with a glass/glass homogenizer. Then the homogenate was diluted 1:4 with nargase-free IM and centrifuged at 2,000 × g for 4 min. After centrifugation, the supernatant was passed through a cheesecloth and centrifuged at 12,000 × g for 9 min. To permeabilize the synaptosomes, the resulting pellet was suspended in 10 ml of ice-cold digitonin medium (IM + 0.02% digitonin), transferred to a small glass homogenizer, and manually homogenized 8–10 times to obtain a homogenous suspension. Finally the suspension was centrifuged at 12,000 × g for 11 min, and the resulting pellet was suspended in 400 μl of IM/g of tissue. Respirometry—Mitochondrial respiration was measured with a Clark-type oxygen electrode by means of high resolution respirometry using an OROBOROS Oxygraph-2k (Oroboros, Innsbruck, Austria) (26Gnaiger E. Respir. Phys. 2001; 128: 277-297Crossref PubMed Scopus (211) Google Scholar) at 30 °C. Respiration of mitochondria (0.06 mg of protein/ml) was investigated in EGTA medium (120 mm mannitol, 40 mm MOPS, 5 mm KH2PO4, 60 mm KCl, 5 mm MgCl2, and 0.1 mm EGTA, pH 7.4). Extramitochondrial concentrations of free Ca2+ ((Cafree2+)) were adjusted either by up to six sequential Ca2+ additions (each of 20 μm) or by one single addition of 50 μm Ca2+ into the medium. (Cafree2+) under each condition was verified by Fura-2 measurements as described below. Ca2+ Accumulation Measurements—Ca2+ accumulation into isolated mitochondria (0.25 mg of protein/ml) was monitored fluorimetrically in medium A (120 mm mannitol, 40 mm MOPS, 5 mm KH2PO4, and 60 mm KCl, pH 7.4) containing 0.5 μm Calcium Green-5N (Invitrogen). Measurements were performed in stirred and thermostated (30 °C) cuvettes using a Cary Eclipse fluorimeter (Varian Deutschland GmbH) as described previously (27Gizatullina Z.Z. Chen Y. Zierz S. Gellerich F.N. Biochim. Biophys. Acta. 2005; 706: 98-104Crossref Scopus (22) Google Scholar). For investigating CsA effects, medium A plus 2 mm MgCl2 (medium B) was used. Excitation and emission wavelengths were adjusted at 506 and 532 nm, respectively. Measurement of (Cafree2+) in EGTA Medium—(Cafree2+) in the EGTA medium was measured fluorimetrically with Fura-2 (10 μm) as described previously (28Groden, D. L., and Gvan, Z. Cell Calcium 12, 279–287Google Scholar, 29Uto A. Arai H. Ogawa Y. Cell Calcium. 1991; 12: 29-37Crossref PubMed Scopus (100) Google Scholar). Because the EGTA medium contained 5 mm Mg2+ that competitively binds Fura-2 (29Uto A. Arai H. Ogawa Y. Cell Calcium. 1991; 12: 29-37Crossref PubMed Scopus (100) Google Scholar), the dissociation constant (Kd) of the Ca2+-Fura-2 complex was measured experimentally under these conditions 3C. Tanne and F. N. Gellerich, unpublished observation. and found to be 0.3 μm, which was similar to that found in a previous study (28Groden, D. L., and Gvan, Z. Cell Calcium 12, 279–287Google Scholar). ΔΨ Measurements—Mitochondrial ΔΨ was monitored fluorimetrically by the release of safranine (30Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (669) Google Scholar). Fluorescence was measured at 495 nm excitation and 586 nm emission using a Cary Eclipse fluorimeter (Varian) with 10 μm safranine in stirred and thermostated cuvettes (30 °C). Measurements were performed in medium A using mitochondria adjusted to 0.25 mg of protein/ml. Protein Determination—Mitochondrial protein concentrations were determined by the bicinchoninic acid assay (31Wiechelman K.J. Braun R.D. Fitzpatrick J.D. Anal. Biochem. 1988; 175: 231-237Crossref PubMed Scopus (633) Google Scholar). BSA was used as standard. Statistical Analysis—All results are presented as mean ± S.D. or mean ± S.E. as indicated. Statistical significance was analyzed by paired and unpaired two-tailed t test or Bonferroni's test. Stimulation of Glutamate-dependent Respiration by Extramitochondrial Ca2+—To investigate the Ca2+ dependence of oxidative phosphorylation, a model system allowing the stepwise increase of (Cafree2+) from nm to μm concentration ranges was developed. We used an incubation medium containing 100 μm EGTA to keep (Cafree2+) negligibly low (EGTA medium). Fig. 1A shows a respirogram of brain mitochondria isolated from normal rats using 10 mm glutamate and 2 mm malate as substrates. After addition of 2.5 mm ADP, an unusually low glutamate-dependent respiration rate (state 3glu/mal) was obtained. To clarify whether such a modest respiration resulted from an insufficient complex I-related metabolism, we measured the complex II-dependent respiration by applying the specific complex I inhibitor rotenone and succinate as complex II-specific substrate (state 3suc respiration). As shown in Fig. 1A, succinate caused a normal state 3suc respiration that clearly exceeded the state 3glu/mal respiration. This finding suggests an inadequate complex I-mediated respiration. However, if the same experiment was performed in the presence of 1.35 μm (Cafree2+) (Fig. 1B), the state 3glu/mal respiration was more than doubled compared with that in Ca2+ free conditions (Fig. 1, A and B). Thus, the increase in (Cafree2+) induced a normalization of the mitochondrial complex I-dependent respiration in normal brain mitochondria. In a second approach, state 3glu/mal respiration was titrated by sequential Ca2+ additions (Fig. 1C). Again state 3glu/mal was very low under Ca2+ free conditions but increased stepwisely until (Cafree2+) reached 2 μm. At this (Cafree2+) concentration, the maximum state 3glu/mal respiration was observed, whereas further Ca2+ additions provoked an inhibitory effect. When Ca2+-induced respiration rates were normalized against the corresponding respiration rate under Ca2+-free conditions and plotted versus (Cafree2+) (Fig. 1F), about a 2-fold increase of state 3glu/mal respiration by 2 μm (Cafree2+) was revealed. To further ascertain whether the pronounced Ca2+ stimulation of state 3glu/mal respiration is a characteristic property of the glutamate metabolism, we investigated the substrate dependence of this effect in more detail. In particular, we considered pyruvate/malate as an alternative substrate of complex I-dependent respiration. We found that state 3pyr/mal respiration was also significantly activated (16 ± 3%) but to a much lesser extent than state 3glu/mal (87 ± 8%; Fig. 1, E and F). Additional experiments revealed that the activation of state 3pyr/mal is not caused by extramitochondrial Ca2+ but is a result of large time requirement for complete activation of pyruvate dehydrogenase. State 3suc respiration remained unaffected by (Cafree2+) (Fig. 1D). Thus, glutamate/malate-dependent respiration could be identified as the most sensitive target of (Cafree2+). The (Cafree2+) concentration required for half-maximum activation of state 3glu/mal respiration was 0.26 ± 0.02 μm. However, this value was much lower than the Km calculated for mitochondrial Ca2+ accumulation via the Ca2+ uniporter under similar conditions 4A. Knabe and F. N. Gellerich, unpublished observation. (2.5 ± 0.2 μm). This finding suggests that Ca2+-dependent activation of state 3glu/mal respiration cannot be mediated by the mitochondrial Ca2+ uniporter. If this assumption is valid, state 3glu/mal respiration should also be activated by (Cafree2+) in the presence of RR, an inhibitor of the Ca2+ uniporter. Indeed 250 nm RR, a dose able to block mitochondrial Ca2+ uptake through the Ca2+ uniporter completely under the conditions used here (not shown), did not prevent the (Cafree2+)-dependent state 3glu/mal activation (Fig. 1F). Thus, mechanisms underlying the stimulation of glutamate/malate-dependent respiration do not require intramitochondrial Ca2+ accumulation and therefore must be initiated outside the Ca2+-impermeable mitochondrial inner membrane. Glutamate is taken up by mitochondria either via aralar, leading to its subsequent transamination by aspartate aminotransferase (inhibitable by aminooxyacetate (AOA)), or via the glutamate/OH carrier followed by its desamination by glutamate dehydrogenase (19Palmieri L. Pardo B. Lasorsa F.M. del Arco A. Kobayashi K. Iijima M. Runswick M.J. Walker J.E. Saheki T. Satrustegui J. Palmieri F. EMBO J. 2001; 20: 5060-5069Crossref PubMed Scopus (392) Google Scholar, 32Schoolwerth A.C. Nazar B.L. LaNoue K.F. J. Biol. Chem. 1978; 253: 6177-6183Abstract Full Text PDF PubMed Google Scholar, 33Schoolwerth A.C. Hoover W.J. Daniel C.H. Lanue K.F. Int. J. Biochem. 1980; 12: 145-149Crossref PubMed Scopus (7) Google Scholar, 34Hoek J.B. Coll K.E. Williamson J.R. J. Biol. Chem. 1983; 258: 54-58Abstract Full Text PDF PubMed Google Scholar). However, the activity of the glutamate/OH carrier is low in most organs except liver and kidney (34Hoek J.B. Coll K.E. Williamson J.R. J. Biol. Chem. 1983; 258: 54-58Abstract Full Text PDF PubMed Google Scholar). If so, Ca2+ activation of state 3glu/mal respiration should be inhibited by AOA. Indeed the Ca2+-dependent state 3glu/mal activation was significantly suppressed by 2 mm AOA 5K. Herrmann and F. N. Gellerich, unpublished observation. (-55 ± 5%), confirming the involvement of the aralar/transaminase pathway in the Ca2+ activation. In addition, we found that AOA (250 nm) did not affect the mitochondrial respiration with pyruvate/malate as substrates 5K. Herrmann and F. N. Gellerich, unpublished observation. (not shown). It is therefore likely that activation of state 3glu/mal respiration by extramitochondrial (Cafree2+) is mediated by an enhanced glutamate transport into the mitochondrial matrix via aralar. However, the functional relationship between (Cafree2+) and state 3glu/mal respiration rate exhibited a biphasic nature. At 6.3 μm or higher (Cafree2+), a marked decrease in state 3glu/mal respiration was monitored (Fig. 1, C and F). Because this change was significantly attenuated by 1 μm CsA (data not shown), it was most likely caused by partial opening of the PT pore due to an intramitochondrial Ca2+ overload. This conclusion was further validated by the following finding. In contrast to the Ca2+-dependent activation
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