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• W2053444468 abstract "Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate α-ketoglutarate (αKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that αKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in αKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of αKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo. Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate α-ketoglutarate (αKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that αKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in αKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of αKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo. Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter (CaU) 2The abbreviations used are: CaU, calcium uniporter; αKG, αketoglutarate; αKGDH, αketoglutarate dehydrogenase; AGC, aspartate-glutamate carrier; AOAA, aminooxyacetate; GDH, glutamate dehydrogenase; MAS, malate-aspartate shuttle; mitDH, mitochondrial dehydrogenases; mitGPDH, mitochondrial glycerol-3-phosphate dehydrogenase; NCX, mitochondrial Na+/Ca2+ exchanger; OGC, oxoglutarate carrier; RR, ruthenium red; TMRM, tetramethylrhodamine methyl ester. and activation of mitochondrial dehydrogenases (mitDH) (1Nichols B.J. Denton R.M. Mol. Cell. Biochem. 1995; 149–150: 203-212Crossref PubMed Scopus (71) Google Scholar). However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers (AGCs) and not the CaU (2Lasorsa F.M. Pinton P. Palmieri L. Fiermonte G. Rizzuto R. Palmieri F. J. Biol. Chem. 2003; 278: 38686-38692Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 3Palmieri 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 (399) Google Scholar, 4Pardo 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 (95) Google Scholar, 5Satrústegui J. Pardo B. del Arco A. Physiol. Rev. 2007; 87: 29-67Crossref PubMed Scopus (205) Google Scholar). Aralar (Slc25a12), also named aralar1, the AGC isoform with predominant expression in brain (6Begum L. Jalil M.A. Kobayashi K. Iijima M. Li M.X. Yasuda T. Horiuchi M. del Arco A. Satrustegui J. Saheki T. Biochim. Biophys. Acta. 2002; 1574: 283-292Crossref PubMed Scopus (55) Google Scholar, 7del Arco A. Morcillo J. Martinez-Morales J.R. Galian C. Martos V. Bovolenta P. Satrustegui J. Eur. J. Biochem. 2002; 269: 3313-3320Crossref PubMed Scopus (66) Google Scholar, 8Ramos M. del Arco A. Pardo B. Martinez-Serrano A. Martinez-Morales J.R. Kobayashi K. Yasuda T. Bogonez E. Bovolenta P. Saheki T. Satrustegui J. Brain Res. Dev. Brain Res. 2003; 143: 33-46Crossref PubMed Scopus (129) Google Scholar), is a component of the NADH malate-aspartate shuttle (MAS), which in brain is activated by extramitochondrial Ca2+ (S0.5 324 nm) (4Pardo 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 (95) Google Scholar). Immunocytochemistry and in situ hybridization data and mRNA levels in acutely isolated brain cells indicate that aralar is localized preferentially in neurons (8Ramos M. del Arco A. Pardo B. Martinez-Serrano A. Martinez-Morales J.R. Kobayashi K. Yasuda T. Bogonez E. Bovolenta P. Saheki T. Satrustegui J. Brain Res. Dev. Brain Res. 2003; 143: 33-46Crossref PubMed Scopus (129) Google Scholar, 9Berkich D.A. Ola M.S. Cole J. Sweatt A.J. Hutson S.M. LaNoue K.F. J. Neurosci. Res. 2007; 85: 3367-3377Crossref PubMed Scopus (87) Google Scholar, 10Cahoy J.D. Emery B. Kaushal A. Foo L.C. Zamanian J.L. Christopherson K.S. Xing Y. Lubischer J.L. Krieg P.A. Krupenko S.A. Thompson W.J. Barres B.A. J. Neurosci. 2008; 28: 264-278Crossref PubMed Scopus (2276) Google Scholar, 11Xu Y. Ola M.S. Berkich D.A. Gardner T.W. Barber A.J. Palmieri F. Hutson S.M. LaNoue K.F. J. Neurochem. 2007; 101: 120-131Crossref PubMed Scopus (56) Google Scholar, 12Lovatt D. Sonnewald U. Waagepetersen H.S. Schousboe A. He W. Lin J.H. Han X. Takano T. Wang S. Sim F.J. Goldman S.A. Nedergaard M. J. Neurosci. 2007; 27: 12255-12266Crossref PubMed Scopus (373) Google Scholar). This is consistent with a higher MAS activity in neuronal than astrocyte cultures (8Ramos M. del Arco A. Pardo B. Martinez-Serrano A. Martinez-Morales J.R. Kobayashi K. Yasuda T. Bogonez E. Bovolenta P. Saheki T. Satrustegui J. Brain Res. Dev. Brain Res. 2003; 143: 33-46Crossref PubMed Scopus (129) Google Scholar) and with aralar being one of the more enriched proteins during differentiation of P19 cells to a neuronal phenotype (13Watkins J. Basu S. Bogenhagen D.F. J. Proteome Res. 2008; 7: 328-338Crossref PubMed Scopus (22) Google Scholar). It is also consistent with the higher levels of aralar in total than in synaptosome-free mitochondrial fractions (9Berkich D.A. Ola M.S. Cole J. Sweatt A.J. Hutson S.M. LaNoue K.F. J. Neurosci. Res. 2007; 85: 3367-3377Crossref PubMed Scopus (87) Google Scholar). Studies in cultured neurons, which have aralar as only AGC isoform, showed that small Ca2+ signals that have limited access to mitochondria are able to activate the aralar-MAS pathway (4Pardo 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 (95) Google Scholar). However, large Ca2+ signals that induce robust mitochondrial Ca2+ transients fail to activate the pathway (4Pardo 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 (95) Google Scholar). This suggested that in neuronal mitochondria the aralar-MAS pathway is inhibited under conditions in which the CaU-mitDH pathway is activated. This surprising result indicates that Ca2+ activation of the malate aspartate shuttle and tricarboxylic acid cycle activity are somehow mutually exclusive in neurons. We have now studied the interplay between the AGG-MAS and CaU-mitDH pathways in brain mitochondria under Ca2+-stimulation conditions. Our results show that the shared metabolite αKG controls the relationship between the second transporter of MAS, the oxoglutarate carrier (OGC, Slc25a11), and αKGDH in the Krebs cycle, by virtue of the effects of Ca2+ on the kinetics of αKGDH. Interestingly, the inhibition of OGC and MAS is fully reversible, in parallel with Ca2+ egress from mitochondria. Behaviorally evoked brain activation results in an increased cerebral blood flow and increased glucose utilization, but paradoxically, this is not accompanied with an equivalent increase in oxygen utilization (14Fox P.T. Raichle M.E. Mintun M.A. Dence C. Science. 1988; 241: 462-464Crossref PubMed Scopus (1336) Google Scholar, 15Raichle M.E. Mintun M.A. Annu. Rev. Neurosci. 2006; 29: 449-476Crossref PubMed Scopus (1158) Google Scholar). As a consequence, the oxygen glucose index, which is close to 6 when glucose is fully oxidized in resting conditions, falls to about 5 (16Madsen P.L. Cruz N.F. Sokoloff L. Dienel G.A. J. Cereb. Blood Flow Metab. 1999; 19: 393-400Crossref PubMed Scopus (143) Google Scholar). This is accompanied by an increase in brain lactate production (14Fox P.T. Raichle M.E. Mintun M.A. Dence C. Science. 1988; 241: 462-464Crossref PubMed Scopus (1336) Google Scholar, 16Madsen P.L. Cruz N.F. Sokoloff L. Dienel G.A. J. Cereb. Blood Flow Metab. 1999; 19: 393-400Crossref PubMed Scopus (143) Google Scholar, 17Cruz N.F. Ball K.K. Dienel G.A. J. Neurosci. Res. 2007; 85: 3254-3266Crossref PubMed Scopus (59) Google Scholar, 18Dienel G.A. Wang R.Y. Cruz N.F. J. Cereb. Blood Flow Metab. 2002; 22: 1490-1502Crossref PubMed Google Scholar, 19Mangia S. Tkac I. Gruetter R. Van de Moortele P.F. Maraviglia B. Ugurbil K. J. Cereb. Blood Flow Metab. 2007; 27: 1055-1063Crossref PubMed Scopus (221) Google Scholar). Our results suggest that MAS inhibition during Ca2+-induced Krebs cycle activation would drive pyruvate to lactate formation and may play a role in lactate formation during brain activation. Animals and Materials-3-Month-old C57BL/6xSv129 mice were housed with a 12-h light cycle and fed ad libitum on standard chow. Animals were sacrificed by cervical dislocation, and the tissue of interest was quickly dissected and kept in ice-cold media for mitochondrial isolation, which was carried out at 4 °C. All animal procedures were approved by European guidelines. All reagents were obtained from Sigma, except malate dehydrogenase (Roche Applied Science), Fura-2, Calcium-Green5N (Molecular Probes, Eugene, OR), and CGP-37157 (Tocris Biosciences, Ellisville, MO). Mitochondrial Isolation-Mitochondria were isolated from the brain, liver, and heart of 3-month-old C57BL/6xSv129 mice, as described previously (20Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 21Martinez-Serrano A. Satrustegui J. Mol. Biol. Cell. 1992; 3: 235-248Crossref PubMed Scopus (29) Google Scholar, 22Rolfe D.F. Hulbert A.J. Brand M.D. Biochim. Biophys. Acta. 1994; 1188: 405-416Crossref PubMed Scopus (226) Google Scholar) with modifications (4Pardo 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 (95) Google Scholar, 23Jalil M.A. Begum L. Contreras L. Pardo B. Iijima M. Li M.X. Ramos M. Marmol P. Horiuchi M. Shimotsu K. Nakagawa S. Okubo A. Sameshima M. Isashiki Y. Del Arco A. Kobayashi K. Satrustegui J. Saheki T. J. Biol. Chem. 2005; 280: 31333-31339Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and were kept on ice in MSK (in mm: 75 mannitol, 25 sucrose, 5 potassium phosphate, 20 Tris-HCl, 0.5 EDTA, 100 KCl, and 0.1% bovine serum albumin, pH 7.4). Mitochondrial protein was measured by the Bradford method, with bovine serum albumin as standard. MAS Reconstitution and mitGPDH Measurement-MAS was reconstituted following published methods (24Atlante A. Gagliardi S. Marra E. Calissano P. Passarella S. J. Neurochem. 1999; 73: 237-246Crossref PubMed Scopus (57) Google Scholar, 25Cederbaum A.I. Lieber C.S. Beattie D.S. Rubin E. Arch. Biochem. Biophys. 1973; 158: 763-781Crossref PubMed Scopus (70) Google Scholar, 26Cheeseman A.J. Clark J.B. J. Neurochem. 1988; 50: 1559-1565Crossref PubMed Scopus (83) Google Scholar), with modifications (4Pardo 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 (95) Google Scholar, 23Jalil M.A. Begum L. Contreras L. Pardo B. Iijima M. Li M.X. Ramos M. Marmol P. Horiuchi M. Shimotsu K. Nakagawa S. Okubo A. Sameshima M. Isashiki Y. Del Arco A. Kobayashi K. Satrustegui J. Saheki T. J. Biol. Chem. 2005; 280: 31333-31339Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Briefly, mitochondria (0.1–0.15 mg of brain and liver and 0.020–0.030 mg of heart) were resuspended in 3 ml of MSK(with 100 μm digitonin for brain preparations), and the shuttle was reconstituted in the presence of 4 units/ml aspartate aminotransferase, 6 units/ml malate dehydrogenase, 66 μm NADH, 5 mm aspartate, 5 mm malate, 0.5 mm ADP. When appropriate, 200 nm Ruthenium Red (RR) and calibrated calcium concentrations were present. mitGPDH activity was measured in a similar way in MSK, with 0.5 mm ADP, 66 μm NADH, and 2 units/ml glycerol-3-phosphate dehydrogenase (27Dawson A.G. Cooney G.J. FEBS Lett. 1978; 91: 169-172Crossref PubMed Scopus (13) Google Scholar). Once a base line is achieved, either 5 mm glutamate (MAS) or 5 mm glycerol 3-phosphate (mitGPDH) was added to trigger shuttle activity, coupled to the decrease in NADH fluorescence (excitation 340 nm, excitation 465 nm). All assays were performed at 37 °C under constant stirring. Measurement of OGC Activity-Transport of α-KG through the OGC was measured by a modification of the MAS reconstitution system. Appropriate amounts of mitochondrial protein of the tissue of interest were resuspended in 3 ml of MSK supplemented with 66 μm NADH, 0.5 mm ADP, 3 mm (NH4)2SO4, 5 mm glutamate, 10 units/ml GDH (and 100 μm digitonin when brain mitochondria were assayed). 5 mm malate addition initiated αKG efflux from mitochondria, which was followed by a decrease in NADH fluorescence. Experiments were performed at 37 °C under constant stirring. When indicated, 200 nm RR and calibrated calcium additions were made. Free Calcium Calibration-The free calcium concentrations in the assays obtained after the different CaCl2 additions were determined in the presence of Fura-2 (under 1 μm, Kd = 224 nm; excitation, 340 and 380 nm; emission, 510 nm; concentration 5 μm) or Calcium-Green5N (over 1 μm, Kd = 14 μm; excitation, 506 nm; emission, 532 nm; concentration 0.1 μm). Free calcium concentration was calculated as established for ratiometric and nonratiometric probes (28Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 29Martinez A. Vitorica J. Satrustegui J. Neurosci. Lett. 1988; 88: 336-342Crossref PubMed Scopus (65) Google Scholar). Enzymatic Assays-Enzyme activities were assayed in either freshly isolated or freeze-thawed mitochondria from different tissues of 3-month-old C57BL/6xSv129 mice. Experiments were performed in a final volume of 200 μl and 20–50 μg of protein in a FLUOstar optima (BMG-Labtech) plate fluorimeter at 30 °C. Activities were calculated as the initial slope of NADH fluorescence changes per mg of protein. An NADH calibration curve was performed daily by adding known amounts of NADH to medium. The following additions were made to the medium (MME: 50 mm KCl, 10 mm HEPES, pH 7.4; 0.2% Triton, and 10 μm rotenone (32Starkov A.A. Fiskum G. Chinopoulos C. Lorenzo B.J. Browne S.E. Patel M.S. Beal M.F. J. Neurosci. 2004; 24: 7779-7788Crossref PubMed Scopus (561) Google Scholar): 0.2 mm NAD+ for measurement of GDH; 0.2 mm NADH, 5 units/ml malate dehydrogenase, and 12.5 mm αKG for aspartate aminotransferase measurement; 0.2 mm NAD+, 0.3 mm thiamine pyrophosphate, 10 μm CaCl2, 0.2 mm MgCl2, 0.14 mm CoASH, and 0.5 mm ADP to assay αKGDH. After establishment of a base line, activity was triggered with the appropriate substrate as follows: 5 mm glutamate or aspartate and 12.5 mm αKG (GDH, aspartate aminotransferase, and αKGDH, respectively (30Robert R. Horder M. Bergmeyer H.U. Bergmeyer J. Grassl M. Methods of Enzymatic Analysis. Enzymes 1: Oxidoreductases, Transferases. 3rd Ed. Vol. III. Verlag Chemie, Weinheim, Germany1983Google Scholar, 31Erakovic V. Zupan G. Varljen J. Laginja J. Simonic A. Epilepsia. 2001; 42: 181-189Crossref PubMed Scopus (33) Google Scholar, 32Starkov A.A. Fiskum G. Chinopoulos C. Lorenzo B.J. Browne S.E. Patel M.S. Beal M.F. J. Neurosci. 2004; 24: 7779-7788Crossref PubMed Scopus (561) Google Scholar, 33Tretter L. Adam-Vizi V. J. Neurosci. 2000; 20: 8972-8979Crossref PubMed Google Scholar). Mitochondrial Membrane Potential-Mitochondrial membrane potential was estimated by quenching of tetramethylrhodamine methyl ester (TMRM, 549 nm excitation, 575 nm emission) fluorescence, as described previously (34Chalmers S. Nicholls D.G. J. Biol. Chem. 2003; 278: 19062-19070Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Briefly, after stabilization of TMRM (300 nm) fluorescence, 100 μg of mitochondria were added to 2 ml of MSK containing 5 mm aspartate. Changes in membrane potential were followed qualitatively by the variations of the TMRM fluorescence after addition of substrates (glutamate/malate, 5 mm), ADP (0.5 mm), and calcium (5 μm free calcium). When indicated, 50 mm Na+ replaced 50 mm K+ in MSK (MSKNa medium). A calcium unbuffered medium (MSK or MSKNa without EDTA) was also used. Ca2+ Egress from Mitochondria-Brain mitochondria (0.25 mg/ml) were incubated in calcium-unbuffered medium (i.e. MSK or MSKNa without EDTA) in the presence of ADP (0.5 mm), aspartate (5 mm), malate (5 mm), digitonin (100 μm) and Calcium-Green5N (0.1 μm). After a stable base line was achieved, an addition of 10 μm CaCl2 was made (40 nmol/mg, giving a free calcium of 5 μm), and calcium uptake was monitored for 5 min before glutamate (5 mm) addition. Where indicated, ruthenium red (200 nm) was added to stop calcium uptake through the uniporter. In some assays, CGP-37157 (10 μm) was added to inhibit the mitochondrial Na+-Ca2+ exchanger (NCX) (35Hernandez-SanMiguel E. Vay L. Santo-Domingo J. Lobaton C.D. Moreno A. Montero M. Alvarez J. Cell Calcium. 2006; 40: 53-61Crossref PubMed Scopus (55) Google Scholar). Regulation of MAS and Glycerol-3-P Dehydrogenase Activity by Extramitochondrial and Intramitochondrial Ca2+-MAS activity was shown to increase in response to extramitochondrial calcium in brain and heart mitochondria (3Palmieri 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 (399) Google Scholar, 4Pardo 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 (95) Google Scholar, 36Contreras L. Gomez-Puertas P. Iijima M. Kobayashi K. Saheki T. Satrustegui J. J. Biol. Chem. 2007; 282: 7098-7106Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Paradoxically, it became inhibited in a calcium-dependent manner when calcium was allowed to enter the mitochondria through the Ca2+ uniporter (4Pardo 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 (95) Google Scholar). In brain mitochondria, the remaining activity drops to 79 ± 4%, 58 ± 7%, and 17 ± 3% in the presence of 0, 0.3, and 5 μm [Ca2+]free, respectively (Fig. 1A). MAS inhibition by intramitochondrial Ca2+ is not as large in heart mitochondria, with 77 ± 6% residual activity at 5 μm free calcium (Fig. 1B). In contrast, MAS activity in liver mitochondria is not affected by intramitochondrial Ca2+, as its activity is the same in the absence or presence of RR, although it is activated by extramitochondrial calcium to a smaller extent than in brain or heart (Fig. 1C) as reported earlier (36Contreras L. Gomez-Puertas P. Iijima M. Kobayashi K. Saheki T. Satrustegui J. J. Biol. Chem. 2007; 282: 7098-7106Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It is unlikely that the inhibitory effects of matrix Ca2+ on MAS activity are because of Ca2+-induced permeability transition pore opening and loss of mitochondrial metabolites (particularly NAD+) as follows: first, because assays were conducted in the presence of 0.5 mm ADP, which is a known inhibitor of permeability transition pore opening (37Bernardi P. Krauskopf A. Basso E. Petronilli V. Blachly-Dyson E. Di Lisa F. Forte M.A. FEBS J. 2006; 273: 2077-2099Crossref PubMed Scopus (558) Google Scholar); second, because inhibition of MAS by Ca2+ matrix was not observed in liver mitochondria (Fig. 1C), which retains half the amount of Ca2+ than brain mitochondria before undergoing permeability transition (38Panov A. Dikalov S. Shalbuyeva N. Hemendinger R. Greenamyre J.T. Rosenfeld J. Am. J. Physiol. 2007; 292: C708-C718Crossref PubMed Scopus (109) Google Scholar); and third, because inhibition of MAS by matrix Ca2+ in brain and heart mitochondria is still observed in the presence of 5 μm cyclosporin A (results not shown). We also investigated the effect of extra- and intramitochondrial Ca2+ on mitochondrial glycerol-3-phosphate dehydrogenase (mitGPDH), the mitochondrial component of the glycerol 3-phosphate shuttle. The activity of this enzyme is known to be activated by low concentrations of extramitochondrial calcium (≈35–100 nm (39Rutter G. Pralong W. Wollheim C. Biochim. Biophys. Acta. 1992; 1175: 107-113Crossref PubMed Scopus (70) Google Scholar) through a decrease in the Km value for glycerol 3-phosphate of the mitGPDH (40MacDonald M.J. Brown L.J. Arch. Biochem. Biophys. 1996; 326: 79-84Crossref PubMed Scopus (72) Google Scholar, 41Wernette M.E. Ochs R.S. Lardy H.A. J. Biol. Chem. 1981; 256: 12767-12771Abstract Full Text PDF PubMed Google Scholar). Unlike MAS, mitGPDH was completely Ca2+-dependent, with no detectable activity in the total absence of free Ca2+ in brain and liver mitochondria (supplemental Fig. 1). The activity of mitGPDH in heart mitochondria was not detectable under the conditions of this study, in agreement with the data of Scholz et al. (42Scholz T.D. Koppenhafer S.L. Pediatr. Res. 1995; 38: 221-227Crossref PubMed Scopus (29) Google Scholar, 43Scholz T.D. Koppenhafer S.L. TenEyck C.J. Schutte B.C. J. Mol. Cell. Cardiol. 1997; 29: 1605-1613Abstract Full Text PDF PubMed Scopus (20) Google Scholar) who found a marked decrease of glycerol 3-phosphate shuttle activity in the heart with development. The enzyme requires Ca2+ in the intermembrane space, as full activation is obtained in the presence of RR in all tissues. The kinetics of activation agrees with a low S0.5 for activation, as the maximal activity was attained at 0.3 μm free Ca2+ (supplemental Fig. 1). On the other hand, Ca2+ entry in the matrix (i.e. RR not present in the assay) did not affect mitGPDH activity in either brain or liver mitochondria (supplemental Fig. 1). Therefore, the results indicate that in brain mitochondria Ca2+ activation of MAS, but not mitGPDH, is prevented by strong Ca2+ signals that reach mitochondrial matrix. This suggests that reducing equivalents transfer to mitochondria may still proceed through mitGPDH when Ca2+ entry in mitochondria leads to an inhibition of MAS activity. This depends on whether a glycerol-P shuttle is actually present in brain, with its two enzymes within the same cell, as suggested to be the case from the astrocyte and neuronal transcriptome findings (12Lovatt D. Sonnewald U. Waagepetersen H.S. Schousboe A. He W. Lin J.H. Han X. Takano T. Wang S. Sim F.J. Goldman S.A. Nedergaard M. J. Neurosci. 2007; 27: 12255-12266Crossref PubMed Scopus (373) Google Scholar, 44Leveille P.J. McGinnis J.F. Maxwell D.S. de Vellis J. Brain Res. 1980; 196: 287-305Crossref PubMed Scopus (106) Google Scholar, 45McKenna M.C. Waagepetersen H.S. Schousboe A. Sonnewald U. Biochem. Pharmacol. 2006; 71: 399-407Crossref PubMed Scopus (241) Google Scholar, 46Nguyen N.H. Brathe A. Hassel B. J. Neurochem. 2003; 85: 831-842Crossref PubMed Scopus (75) Google Scholar). Effect of Extra- and Intramitochondrial Calcium on OGC Activity-The activity of the OGC was studied by measuring the efflux of αKG from mitochondria, in medium containing glutamate dehydrogenase (GDH). GDH will transform αKG into glutamate in the presence of NADH and ammonium, resulting in a decrease in NADH fluorescence, when malate is added to trigger αKG efflux from mitochondria (supplemental Fig. 2). The supplemental Fig. 2B shows that the decrease of NADH fluorescence was dependent on the presence of ammonium and was prevented by the OGC inhibitor phenylsuccinate (45McKenna M.C. Waagepetersen H.S. Schousboe A. Sonnewald U. Biochem. Pharmacol. 2006; 71: 399-407Crossref PubMed Scopus (241) Google Scholar, 47de Bari L. Atlante A. Guaragnella N. Principato G. Passarella S. Biochem. J. 2002; 365: 391-403Crossref PubMed Scopus (60) Google Scholar). To test the effects of both extra- and intramitochondrial calcium on OGC activity, αKG efflux was measured in the absence or presence of the calcium uniporter inhibitor RR, both in Ca2+-free medium or in the presence of 5 μm free Ca2+. In the presence of 200 nm RR, there was no difference in αKG efflux at the two calcium concentrations in brain, heart and liver mitochondria (Table 1), indicating that the OGC is not activated by extramitochondrial Ca2+, in accordance to the lack of calcium-binding motifs in the OGC sequence (48Runswick M. Walker J. Bisaccia F. Iacobazzi V. Palmieri F. Biochemistry. 1990; 29: 11033-11040Crossref PubMed Scopus (141) Google Scholar).TABLE 1Effect of intra- and extramitochondrial calcium on the activity of the OGC αKG efflux was measured in brain, heart, and liver mitochondria from 3-month-old C57BL/6xSv129 mice at 0 (i.e. under the Fura2 limit detection, <20 nm) and 5 μm free calcium, in the presence (RR present) or absence (No RR) of 200 nm RR. Data are mean ± S.E. of 4–6 independent experiments performed in duplicate. Asterisks denote significant difference (Student’s t test) between OGC activities in presence or absence of RR at each calcium concentration (*, p < 0.05; **, p < 0.005; ***, p < 0.0005).OGC activity0 μm [Ca2+]free5 μm [Ca2+]freeNo RRRR presentNo RRRR presentnmol of NADH min–1 mg protein–1Brain116.03 ± 33.12105.16 ± 1746.06 ± 7***127.5 ± 15Heart378.13 ± 66329.2 ± 56356.4 ± 66*447.4 ± 53Liver66.87 ± 19*101.2 ± 2844.6 ± 8**103.2 ± 11 Open table in a new tab Having shown that extramitochondrial Ca2+ does not modify the OGC activity, the influence of intramitochondrial Ca2+ was studied by carrying out the same experiments in the absence of RR, when the Ca2+ uniporter is not inhibited (Table 1). Under these conditions the addition of Ca2+ results in a decrease of αKG efflux in mitochondria from brain and heart (to 37 ± 5% and 78 ± 10% residual activity, respectively (Table 1)), indicating that the OGC in these tissues is inhibited by intramitochondrial Ca2+. In liver, the absence of RR resulted in an inhibition of αKG efflux both in the absence or presence of Ca2+ (Table 1), clearly a nonspecific effect, unrelated to Ca2+ entry in mitochondria, which was not studied any further. Therefore, inhibition of αKG efflux by mitochondrial Ca2+ may explain the inhibition of MAS in brain and heart mitochondria. Influence of Mitochondrial αKG Sources and Utilization Pathways on αKG Efflux Along the OGC-OGC and αKGDH have a common substrate, αKG. When mitochondrial Ca2+ increases, it activates αKGDH, which causes a decrease in the Km value for its substrate, αKG, and a decrease in mitochondrial αKG levels (49McCormack J.G. Denton R.M. Biochem. J. 1979; 180: 533-544Crossref PubMed Scopus (323) Google Scholar, 50Wan B. LaNoue K.F. Cheung J.Y. Scaduto Jr., R.C. J. Biol. Chem. 1989; 264: 13430-13439Abstract Full Text PDF PubMed Google Scholar). Our working hypothesis is that this leads to the decrease in OGC activity that we have shown above in brain and heart mitochondria. As OGC is a member of MAS, this may explain the decrease of MAS activity in brain and heart mitochondria when calcium is allowed to enter the organelle (see Fig. 1A and supplemental Fig. 2A). We figured that a high OGC activity with respect to αKGDH would make the OGC relatively independent of αKGDH activity, and this could be a possible explanation for the lack of effect of intramitochondrial Ca2+ on liver MAS and OGC activities. However, Table 2 shows that this is not the case. Measurements of αKGDH activity in mitochondria from different tissues showed that the ratio of αKG efflux to αKGDH activity is similar in liver and brain mitochondria.TABLE 2Enzyme and transporter activities involved in αKG production and utilization in mitochondria using glutamate and malate as substrates The results are mean ± S.E. of four independent experiments performed at least in duplicate. αKG production was computed as the sum of AGC-MAS and GDH, whereas αKG utilization was the sum of αKGDH and OGC. Differences (Bonferroni test) with liver (*, p < 0.05; **, p < 0.005) or heart (***, p < 0.005) are indicated.ActivityBrainHeartLivernmol of NADH min–1 mg protein–1mitAST1798 ± 3742170 ± 2832569 ± 608αKGDH31.38 ± 6.78***275.4 ± 2821.86 ± 4.5***OGC127.5 ± 16***447.4 ± 59103.2 ± 12***AGC-MA" @default.
• W2053444468 created "2016-06-24" @default.
• W2053444468 creator A5002453287 @default.
• W2053444468 creator A5061848542 @default.
• W2053444468 date "2009-03-01" @default.
• W2053444468 modified "2023-10-17" @default.
• W2053444468 title "Calcium Signaling in Brain Mitochondria" @default.
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• W2053444468 doi "https://doi.org/10.1074/jbc.m808066200" @default.
• W2053444468 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2652317" @default.
• W2053444468 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19129175" @default.
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