FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2049366846> ?p ?o ?g. }

• W2049366846 endingPage "14456" @default.
• W2049366846 startingPage "14448" @default.
• W2049366846 abstract "The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport. The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport. Recently, it has emerged that mitochondrial dysfunctions play an important role in the pathogenesis of degenerative diseases of the central nervous system (1Greenamyre J.T. Sherer T.B. Betarbet R. Panov A.V. IUBMB Life. 2001; 52: 135-141Crossref PubMed Scopus (278) Google Scholar, 2Martin L.J. Liu Z. Chen K. Price A.C. Pan Y. Swaby J.A. Golden W.C. J. Comp. Neurol. 2007; 500: 20-46Crossref PubMed Scopus (230) Google Scholar, 3Kwong J.Q. Beal M.F. Manfredi G. J. Neurochem. 2006; 97: 1659-1675Crossref PubMed Scopus (137) Google Scholar). The processes underlying neuronal degeneration are complex, and some authors suggest that several genetic alterations are involved (4Shaw P.J. J. Neurol. Neurosurg. Psychiatry. 2005; 76: 1046-1057Crossref PubMed Scopus (253) Google Scholar). However, another level of complexity may be derived from the fact that virtually all cellular activities depend upon energy metabolism in the cell (5Erecinska M. Nelson D. J. Neurochem. 1994; 63: 1033-1041Crossref PubMed Scopus (54) Google Scholar). Alterations in energy metabolism processes within cells may also contribute to pathogenic mechanisms underlying neurodegenerative disease. A large body of evidence suggests that increased oxidative stress is an important pathogenic mechanism that promotes neurodegeneration (6Halliwell B. J. Neurochem. 2006; 97: 1634-1658Crossref PubMed Scopus (2077) Google Scholar). Because neurons have a long life span, and most neurodegenerative diseases have a clear association with age (7Bohr V.A. Ottersen O.P. Tonjum T. Neuroscience. 2007; 145: 1183-1186Crossref PubMed Scopus (48) Google Scholar), it is important to understand mechanisms underlying reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; AGC, aspartate-glutamate carrier; BM, brain mitochondria; BSA, bovine serum albumin; CCCP, cyanide-m-chlorophenylhydrazone; α-KG, α-ketoglutarate; α-KGDHC, α-ketoglutarate dehydrogenase complex; MAS, malate aspartate shuttle; MOPS, 4-morpholinepropane sulfonic acid sodium salt; OAA, oxaloacetate; RET, reverse electron transport; SDH, succinate dehydrogenase; TPP+, tetraphenyl phosphonium; RHM, rat heart mitochondria; non-BSA-BM, BM isolated in the absence of BSA. production in neurons. Recently, Kudin et al. (8Kudin A.P. Malinska D. Kunz W.S. Biochim. Biophys. Acta. 2008; 1777: 689-695Crossref PubMed Scopus (70) Google Scholar) analyzed the contribution of mitochondria to the total ROS production in brain tissue. They concluded that mitochondria are the major source of ROS and that at least 50% of ROS generated by brain mitochondria was associated with succinate-supported reverse electron transport (RET). Under conditions of normoxia, about 1% of the respiratory chain electron flow was redirected to form superoxide (8Kudin A.P. Malinska D. Kunz W.S. Biochim. Biophys. Acta. 2008; 1777: 689-695Crossref PubMed Scopus (70) Google Scholar). Recently, we suggested that the organization of the respiratory chain complexes into supercomplexes that occurs in brain mitochondria (BM) (9Schagger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar) may represent one of the intrinsic mechanisms to prevent excessive ROS generation (10Panov A. Dikalov S. Shalbuyeva N. Hemendinger R. Greenamyre J.T. Rosenfeld J. Am. J. Physiol. 2007; 292: C708-C718Crossref PubMed Scopus (109) Google Scholar). In this paper, we put forward the hypothesis that inhibition of Complex II by oxaloacetate (OAA) represents another important intrinsic mechanism to prevent oxidative stress. We provide evidence that glutamate and pyruvate specifically exert control over the production of ROS at the level of Complex II. Below we present a brief account of published theoretical and experimental evidence that underlie our hypothesis. The neural processing of information is metabolically expensive (11Attwell D. Laughlin S.B. J. Cereb. Blood Flow Metab. 2001; 21: 1133-1145Crossref PubMed Scopus (2206) Google Scholar). More than 80% of energy is spent postsynaptically to restore the ionic composition of neurons (11Attwell D. Laughlin S.B. J. Cereb. Blood Flow Metab. 2001; 21: 1133-1145Crossref PubMed Scopus (2206) Google Scholar). When neurons are activated, reuptake of glutamate stimulates aerobic glycolysis in astroglial cells (12Pellerin L. Magistretti P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10625-10629Crossref PubMed Scopus (2097) Google Scholar), thereby making lactate the major substrate for neuronal mitochondria (4Shaw P.J. J. Neurol. Neurosurg. Psychiatry. 2005; 76: 1046-1057Crossref PubMed Scopus (253) Google Scholar, 13Hertz L. Peng L. Dienel G.A. J. Cereb. Blood Flow Metab. 2007; 27: 219-249Crossref PubMed Scopus (439) Google Scholar). However, rapid conversion of lactate to pyruvate in neurons requires activation of the malate-aspartate shuttle (MAS). The shuttle is the major pathway for cytosolic reducing equivalents from NADH to enter the mitochondria and be oxidized (14Berkich D.A. Ola M.S. Cole J. Sweatt A.J. Hutson S.M. LaNoue K.F. J. Neurosci. Res. 2007; 85: 3367-3677Crossref PubMed Scopus (86) Google Scholar, 15McKenna M.C. Waagepetersen H.S. Schousboe A. Sonnewald U. Biochem. Pharmacol. 2006; 71: 399-407Crossref PubMed Scopus (238) Google Scholar). The key component of MAS is the mitochondrial aspartate/glutamate carrier (AGC) (16LaNoue K.F. Schoolwerth A.C. Annu. Rev. Biochem. 1979; 48: 871-922Crossref PubMed Scopus (457) Google Scholar), and recent data suggest that the AGC is expressed mainly in neurons (14Berkich D.A. Ola M.S. Cole J. Sweatt A.J. Hutson S.M. LaNoue K.F. J. Neurosci. Res. 2007; 85: 3367-3677Crossref PubMed Scopus (86) Google Scholar). Absence of the AGC from astrocytes in the brain implies a compartmentation of intermediary metabolism, with glycolysis taking place in astrocytes and lactate oxidation in neurons (13Hertz L. Peng L. Dienel G.A. J. Cereb. Blood Flow Metab. 2007; 27: 219-249Crossref PubMed Scopus (439) Google Scholar, 14Berkich D.A. Ola M.S. Cole J. Sweatt A.J. Hutson S.M. LaNoue K.F. J. Neurosci. Res. 2007; 85: 3367-3677Crossref PubMed Scopus (86) Google Scholar, 17Hertz L. Neurochem. Int. 2004; 45: 285-296Crossref PubMed Scopus (112) Google Scholar). Active operation of MAS requires that a certain amount of glutamate must be transported from synaptic clefts into activated neurons. In isolated BM, it has been shown that besides pyruvate, glutamate is also a good respiratory substrate (5Erecinska M. Nelson D. J. Neurochem. 1994; 63: 1033-1041Crossref PubMed Scopus (54) Google Scholar, 18Yudkoff M. Nelson D. Daikhin Y. Erecinska M. J. Biol. Chem. 1994; 269: 27414-27420Abstract Full Text PDF PubMed Google Scholar). In the presynaptic elements, the concentration of cytosolic glutamate is ∼10 mm at all times (19Kauppinen R.A. Nicholls D.G. Eur. J. Biochem. 1986; 158: 159-165Crossref PubMed Scopus (136) Google Scholar). Yudkoff et al. (18Yudkoff M. Nelson D. Daikhin Y. Erecinska M. J. Biol. Chem. 1994; 269: 27414-27420Abstract Full Text PDF PubMed Google Scholar) have shown that synaptosomal mitochondria utilize glutamate and pyruvate as mitochondrial respiratory substrates. Glutamate is also oxidized by the astroglial mitochondria (13Hertz L. Peng L. Dienel G.A. J. Cereb. Blood Flow Metab. 2007; 27: 219-249Crossref PubMed Scopus (439) Google Scholar). Until recently, it was generally accepted that most of the glutamate is rapidly removed from the synaptic cleft by glutamate transporters EAAT1 and EAAT2 located on presynaptic termini and glial cells (20Rothstein J.D. Martin L. Levey A.I. Dykes-Hoberg M. Jin L. Wu D. Nash N. Kuncl R.W. Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1462) Google Scholar, 21Danbolt N.C. Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3775) Google Scholar, 22Mim C. Balani P. Rauen T. Grewer C. J. Gen. Physiol. 2005; 126: 571-589Crossref PubMed Scopus (72) Google Scholar, 23Torres-Salazar D. Fahlke C. J. Biol. Chem. 2007; 282: 34719-34726Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 24Furness D.N. Dehnes Y. Akhtar A.Q. Rossi D.J. Hamann M. Grutle N.J. Gundersen V. Holmseth S. Lehre K.P. Ullensvang K. Wojewodzic M. Zhou Y. Attwell D. Danbolt N.C. Neuroscience. 2008; 157: 80-94Crossref PubMed Scopus (189) Google Scholar). However, recent data show that a significant fraction of glutamate is rapidly bound and transported by the glutamate transporter isoform, EAAT4, located juxtasynaptically in the membranes of spines and dendrites (20Rothstein J.D. Martin L. Levey A.I. Dykes-Hoberg M. Jin L. Wu D. Nash N. Kuncl R.W. Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1462) Google Scholar, 25Dehnes Y. Chaudhry F.A. Ullensvang K. Lehre K.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1998; 18: 3606-3619Crossref PubMed Google Scholar, 26Kanai Y. Smith C.P. Hediger M.A. FASEB J. 1993; 7: 1450-1459Crossref PubMed Scopus (228) Google Scholar, 27Otis T.S. Kavanaugh M.P. Jahr C.E. Science. 1997; 277: 1515-1518Crossref PubMed Scopus (156) Google Scholar, 28Brasnjo G. Otis T.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6273-6278Crossref PubMed Scopus (28) Google Scholar). At the climbing fiber to Purkinje cell synapses in the cerebellum, about 17% (28Brasnjo G. Otis T.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6273-6278Crossref PubMed Scopus (28) Google Scholar) or more than 50% (29Auger C. Attwell D. Neuron. 2000; 28: 547-558Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) of synaptically released glutamate may be removed by postsynaptic transporters. Besides the cerebellum, EAAT4 protein was found to be omnipresent throughout the fore- and midbrain regions (30Massie A. Cnops L. Smolders I. McCullumsmith R. Kooijman R. Kwak S. Arckens L. Michotte Y. J. Comp. Neurol. 2008; 511: 155-172Crossref PubMed Scopus (49) Google Scholar). Moreover, it was shown that although most of the EAAT2 protein is astroglial, around 15% is distributed in nerve terminals and axons in hippocampal slices and that this protein may be responsible for more than half of the total uptake of glutamate from synaptic clefts (24Furness D.N. Dehnes Y. Akhtar A.Q. Rossi D.J. Hamann M. Grutle N.J. Gundersen V. Holmseth S. Lehre K.P. Ullensvang K. Wojewodzic M. Zhou Y. Attwell D. Danbolt N.C. Neuroscience. 2008; 157: 80-94Crossref PubMed Scopus (189) Google Scholar). These data suggest that postsynaptic transport of glutamate into nerve terminals where mitochondria are located (31Li Z. Okamoto K. Hayashi Y. Sheng M. Cell. 2004; 119: 873-887Abstract Full Text Full Text PDF PubMed Scopus (1129) Google Scholar) may occur in all brain regions. According to calculations of Brasnjo and Otis (28Brasnjo G. Otis T.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6273-6278Crossref PubMed Scopus (28) Google Scholar), in a single synapse, EAAT4 (excitatory amino acid transporter 4) binds and transports postsynaptically about 1.3 ± 0.1 × 106 glutamate molecules. In the brain, on average, 1 mm3 of tissue contains 1 × 108 synapses (32Nicholls D.G. Eur. J. Biochem. 1993; 212: 613-631Crossref PubMed Scopus (207) Google Scholar, 33Abeles M. Corticonics: Neural Circuits of the Cerebral Cortex. Cambridge University Press, Cambridge, UK1991Crossref Google Scholar). Because of the high density of synaptic contacts, the neuronal cells may be exposed to mediators released from hundreds of firing synapses. Thus, in a narrow space of spines and dendrites, several million glutamate molecules postsynaptically transported from synaptic boutons may create local cytosolic concentration of glutamate in the low millimolar range. Consequently, neuronal mitochondria, particularly those located at the axonal or dendritic synaptic junctions, may, in addition to metabolizing pyruvate, temporarily metabolize glutamate and succinate formed during mitochondrial catabolism of γ-aminobutyric acid in postsynaptic cells (34Tillakaratne N.J. Medina-Kauwe L. Gibson K.M. Comp. Biochem. Physiol. A. 1995; 112: 247-263Crossref PubMed Scopus (168) Google Scholar). The purpose of this study was to examine how the neuromediator glutamate affects respiratory activity and ROS generation in nonsynaptic BM when combined with pyruvate and the tricarboxylic acid cycle intermediates succinate and malate. We show that with pyruvate + glutamate + malate, the rate of oxidative phosphorylation increased more than 50%, and in resting mitochondria the rate of ROS generation associated with the reverse electron transport increased severalfold. These effects were observed only with brain and spinal cord mitochondria, not with liver or heart mitochondria, suggesting that they may be restricted to neuronal cells. Taken together, the data presented support the hypothesis that in activated neurons, the neuromediator glutamate stimulates mitochondrial ATP production when energy demand is increased. However, in the absence of energy consumption, glutamate + pyruvate may increase the generation of ROS severalfold. We suggest that intrinsic inhibition of Complex II by oxaloacetate is an important natural protective mechanism against ROS associated with reverse electron transport. Animals—All animal use complied with National Institutes of Health guidelines and was approved by the Institutional Animal Care and Use Committee of the Carolinas Medical Center. Male Sprague-Dawley rats (180–250 g) from Taconic Farms Inc. (Germantown, NY) were used for isolation of BM. Isolation of Mitochondria—Brain mitochondria were isolated from pooled forebrains of three rats using a modified method of Sims (37Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (343) Google Scholar), as described in Ref. 10Panov A. Dikalov S. Shalbuyeva N. Hemendinger R. Greenamyre J.T. Rosenfeld J. Am. J. Physiol. 2007; 292: C708-C718Crossref PubMed Scopus (109) Google Scholar. Rat heart and liver mitochondria were isolated as described in Refs. 10Panov 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 38Panov A. Dikalov S. Shalbueva N. Taylor G. Sherer T. Greenamyer J.T. J. Biol. Chem. 2005; 280: 42026-42035Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar. The isolation medium contained 75 mm mannitol, 150 mm sucrose, 20 mm MOPS, pH 7.2, and 1 mm EGTA. For experiments examining effects of defatted bovine serum albumin (BSA), 0.1% BSA was added to the isolation medium. The final suspensions of mitochondria were prepared using a medium containing 75 mm KCl, 125 mm sucrose, and 10 mm MOPS. Mitochondrial protein was determined with the Pierce Coomassie protein assay reagent kit. Simultaneous Measurement of Mitochondrial Respiration and Membrane Potential—Respiratory activities of the mitochondria were measured using a custom-made plastic minichamber of 560 μl volume equipped with a standard YSI (YSI Inc.) oxygen minielectrode connected to a YSI model 5300 Biological Oxygen Monitor, a custom-made tetraphenylphosphonium (TPP+)-sensitive minielectrode, and a KCl bridge to an Ag/AgCl reference electrode connected to a pH meter. All instruments were connected to the data acquisition system. The incubation medium contained 125 mm KCl, 10 mm MOPS, pH 7.2, 2 mm MgCl2, 2 mm KH2PO4, 10 mm NaCl, 1 mm EGTA, and 0.7 mm CaCl2. At a Ca2+/EGTA ratio of 0.7, the free [Ca2+] is close to 1 μm, as determined using Fura-2. The substrate concentrations were as follows: 5 mm succinate without rotenone, 5 mm glutamate, 2.5 mm pyruvate, 10 mm α-ketoglutarate, and 2 mm malate. Oxidative phosphorylation (state 3) was initiated by the addition of 150 μm ADP. The uncoupled respiration (state 3U) was stimulated by titration with CCCP (0.05 μm aliquots) until the maximum rate of oxygen consumption was obtained. Measurements of Hydrogen Peroxide Generation—H2O2 was measured using the Amplex red (Molecular Probes) method as described in Refs. 10Panov 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 38Panov A. Dikalov S. Shalbueva N. Taylor G. Sherer T. Greenamyer J.T. J. Biol. Chem. 2005; 280: 42026-42035Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar. Respiratory inhibitors and uncoupler were added to the incubation media before the addition of mitochondria. The additions of resorufin or standard solutions of H2O2 (Fluka) were used for calibration of the fluorescence scale. Fluorimetric measurements were made using a highly sensitive fluorometer from C&L Co. (Middletown, PA). Data Acquisition—Data acquisition was performed using hardware and software from C&L Co. Chemicals—Sucrose, mannitol, and other chemicals were from Sigma and of molecular biology grade. All solutions were made using glass bidistilled water. Statistics—Comparisons between two groups were made by unpaired t test, and comparisons between more than two groups were made by analyses of variance followed by post hoc tests. Spontaneous Inhibition of Succinate Oxidation—Nonsynaptic BM are commonly isolated with defatted BSA present either in the isolation medium (8Kudin A.P. Malinska D. Kunz W.S. Biochim. Biophys. Acta. 2008; 1777: 689-695Crossref PubMed Scopus (70) Google Scholar, 10Panov A. Dikalov S. Shalbuyeva N. Hemendinger R. Greenamyre J.T. Rosenfeld J. Am. J. Physiol. 2007; 292: C708-C718Crossref PubMed Scopus (109) Google Scholar) or added to the mitochondrial suspension at some stage of the isolation procedure (37Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (343) Google Scholar). It is believed that BSA binds fatty acids and thus prevents their uncoupling effect on BM (39Tretter L. Mayer-Takacs D. Adam-Vizi V. Neurochem. Int. 2007; 50: 139-147Crossref PubMed Scopus (44) Google Scholar). An important feature of BM from Taconic Sprague-Dawley rats isolated with BSA was that 1–2 h postisolation, BM oxidizing succinate stopped responding to the addition of ADP or the uncoupler CCCP. Simultaneous measurements of respiration and membrane potential (ΔΨ) in BM oxidizing succinate showed that the mitochondria remained depolarized after the initiation of ADP phosphorylation (Fig. 1A). When added to BM with inhibited succinate oxidation, glutamate or pyruvate restored normal response to ADP (Fig. 1A). In contrast, when succinate + glutamate (or pyruvate) were present from the beginning, the state 3 respiration and ΔΨ were not impaired (Fig. 1B). At the same time, oxidation of glutamate + malate or pyruvate + malate by the BSA-BM remained stable even 4–5 h after isolation with high respiratory control ratios (data not shown). The fact that pyruvate and glutamate prevented or abolished inhibition of succinate oxidation suggests that the inhibition was associated with OAA. Because BM isolated in the absence of BSA (non-BSA-BM) showed inhibition of succinate dehydrogenase (Complex II) with practically normal rates of glutamate + malate oxidation (35Panov A. Dikalov S. Rosenfeld J. Free Radic. Biol. Med. 2006; 41: S167Google Scholar), we analyzed interactions between substrates using non-BSA-BM. Effects of Substrate Mixtures on Respiratory Activity of BM—Fig. 2 shows respiratory activities with the non-BSA-BM exposed to various combinations of respiratory substrates. The results were normalized to the rates seen with pyruvate + malate, which was taken as 100%. Fig. 2A shows that the resting respiration rates (state 4) with glutamate + malate and pyruvate + glutamate + malate were the same as with pyruvate + malate. With succinate, succinate + pyruvate, and succinate + glutamate, the state 4 respiratory rates were correspondingly 159% (p < 0.001), 146% (p < 0.001), and 135% (p < 0.05). When malate was also present, the rate of state 4 declined somewhat, and with succinate alone, malate inhibited the state 4 respiration by 43% (p < 0.001) (Fig. 2A). These relatively small effects of malate on the state 4 oxidation of succinate in the presence of pyruvate or glutamate result in significant inhibition of ROS generation, as shown in Figs. 5 and 6.FIGURE 5Glutamate and pyruvate stimulate the succinate supported H2O2 generation only in the non-BSA-brain mitochondria. Incubation conditions were as in Fig. 1. Final volume was 1 ml. Additions were as follows: 5 μm Amplex red, 3 units of horseradish peroxidase, 50 units of superoxide dismutase (Sigma), 0.1 mg of rat brain mitochondria, 5 mm succinate (S), 5 mm glutamate (G), 2.5 mm pyruvate (P), 2 mm malate (M). Malonate (5 mm) was added before BM. Numbers associated with traces show the rates of H2O2 production in pmol of H2O2/min/mg of BM protein. AU, arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6A comparison of the effects of substrate mixtures on the succinate supported state 3 respiration and state 4 generation of H2O2 by non-BSA-brain mitochondria. Incubation conditions were as in Figs. 4 and 5. All experiments were made on the same batch of mitochondria. Data were normalized to the succinate alone, taken as 100%. S, succinate (5 mm); P, pyruvate (2.5 mm); G, glutamate (5 mm); M, malate (2 mm).View Large Image Figure ViewerDownload Hi-res image Download (PPT) When BM oxidized pyruvate + glutamate + malate, the rate of oxidative phosphorylation (state 3) increased by 52% (p < 0.001) (Fig. 2B). With succinate alone, the state 3 respiration was only 44% of the control rate with pyruvate + malate (p < 0.001). With succinate + pyruvate or succinate + pyruvate + malate, the state 3 respiration rates increased to 115 and 106%, respectively (Fig. 2B). The increases were not statistically significant when compared with pyruvate + malate but were significantly higher when compared with succinate alone (p < 0.001; not shown). Glutamate or glutamate + malate added to BM oxidizing succinate also increased the state 3 respiration to the level observed with pyruvate + malate. In BM uncoupled with CCCP, O2 utilization in the presence of pyruvate + glutamate + malate was 71% (p < 0.001) higher than with pyruvate + malate (Fig. 2C). The initial rate of succinate oxidation by uncoupled BM was inhibited by 61% (p < 0.001), and the inhibition rapidly increased. It is likely that the inhibition was caused by OAA, which is consistent with the ability of pyruvate and glutamate to release the inhibition of succinate oxidation (Fig. 2C). With succinate alone, malate slightly inhibited the state 3U respiration (Fig. 2C). Response of Rat Heart Mitochondria to Substrate Mixtures— To study whether the effects of the substrate mixtures on respiratory activities, shown in Fig. 2, are specific for the BM only, we conducted similar experiments with mitochondria from rat heart isolated in the absence of BSA. Fig. 3 shows the rates of respiration in different metabolic states of the rat heart mitochondria (RHM). Unlike BM, the rates of oxygen consumption in the presence of pyruvate + glutamate + malate did not increase in RHM. Oxidation of succinate was significantly lower (p < 0.01) as compared with pyruvate + malate. In contrast to BM, with RHM, the rates of succinate oxidation did not depend on whether the mitochondria were isolated in the presence or absence of BSA. Pyruvate and glutamate added separately (data not shown) or together (Fig. 3, B and C) to the RHM oxidizing succinate increased respiration in metabolic states 3 and 3U to levels associated with pyruvate + malate, and the resting respiration (state 4) increased to a level significantly higher than that seen with pyruvate + malate (p < 0.01) (Fig. 3A). Effects of Substrate Mixtures on ROS Production by BM—We studied the effects of substrate mixtures on ROS generation by the non-BSA-BM (Fig. 4A) or BSA-BM (Fig. 4B) supported by glutamate and pyruvate. Fig. 7 summarizes the results of four separate experiments normalized for ROS generation with pyruvate + malate. Figs. 4A and 7 show that with glutamate + malate, generation of ROS was 4.4-fold lower than with pyruvate + malate and 9-fold lower than with pyruvate + glutamate + malate. When non-BSA-BM simultaneously oxidized pyruvate + glutamate + malate, the rate of ROS generation increased more than 2-fold when compared with pyruvate + malate (Fig. 4A). To analyze whether this increase in ROS production was associated with increased RET, we added malonate, an inhibitor of succinate dehydrogenase (SDH). Fig. 4A shows that the addition of 5 mm malonate inhibited ROS production with pyruvate + glutamate + malate by 77%. Malonate also inhibited ROS generation with pyruvate + malate by 50% (not shown) but had no effect on ROS generation with glutamate + malate or 10 mm α-ketoglutarate (α-KG) (data not shown). CCCP had effects similar to those of malonate on ROS generation with pyruvate + malate or pyruvate + glutamate + malate (data not shown). Fig. 4B shows that with the BSA-BM, ROS generation rates were higher than with the non-BSA-BM, but the pattern of the changes seen with the substrate mixtures described above were the same as those seen with the non-BSA-BM.FIGURE 7Generation of H2O2 by non-BSA rat brain mitochondria oxidizing various substrates and substrate mixtures. Incubation conditions were as in Figs. 4 and 5. The data were normalized to ROS generation with pyruvate + malate (100%). S, succinate (5 mm); P, pyruvate (2.5 mm); G, glutamate (5 mm); M, malate (2 mm).View Large Image Figure ViewerDownload Hi-res image Download (PPT) With succinate alone (Fig. 5), the rate of ROS generation by the BSA-BM was 12-fold higher when compared with the non-BSA-BM. With the BSA-BM, the addition of pyruvate or glutamate to the succinate-oxidizing BM had no effect or caused some inhibition of ROS generation, as compared with succinate alone (data not shown). With the non-BSA-BM, the rate of ROS generation supported by succinate + pyruvate + glutamate + malate was 4-fold higher than with succinate alone (Fig. 5). The addition of malonate or CCCP to the BSA-BM or non-BSA-BM oxidizing succinate alone completely inhibited ROS generation but caused only a partial inhibition with succinate + pyruvate + glutamate + malate with the non-BSA-BM (Fig. 5). Thus, both malonate and CCCP essen" @default.
• W2049366846 created "2016-06-24" @default.
• W2049366846 creator A5000303187 @default.
• W2049366846 creator A5032745219 @default.
• W2049366846 creator A5064178833 @default.
• W2049366846 creator A5065229498 @default.
• W2049366846 creator A5066255279 @default.
• W2049366846 creator A5086468930 @default.
• W2049366846 date "2009-05-01" @default.
• W2049366846 modified "2023-09-30" @default.
• W2049366846 title "The Neuromediator Glutamate, through Specific Substrate Interactions, Enhances Mitochondrial ATP Production and Reactive Oxygen Species Generation in Nonsynaptic Brain Mitochondria" @default.
• W2049366846 cites W1541314725 @default.
• W2049366846 cites W1544076335 @default.
• W2049366846 cites W1558696651 @default.
• W2049366846 cites W1580746914 @default.
• W2049366846 cites W1582272079 @default.
• W2049366846 cites W1942398216 @default.
• W2049366846 cites W1966217930 @default.
• W2049366846 cites W1967192722 @default.
• W2049366846 cites W1969331984 @default.
• W2049366846 cites W1972260106 @default.
• W2049366846 cites W1984168321 @default.
• W2049366846 cites W1994585531 @default.
• W2049366846 cites W1996971085 @default.
• W2049366846 cites W2003410399 @default.
• W2049366846 cites W2005700722 @default.
• W2049366846 cites W2007649442 @default.
• W2049366846 cites W2007815419 @default.
• W2049366846 cites W2012856776 @default.
• W2049366846 cites W2017349018 @default.
• W2049366846 cites W2021460323 @default.
• W2049366846 cites W2025502483 @default.
• W2049366846 cites W2026271507 @default.
• W2049366846 cites W2026796857 @default.
• W2049366846 cites W2027824573 @default.
• W2049366846 cites W2034878114 @default.
• W2049366846 cites W2037749925 @default.
• W2049366846 cites W2042422091 @default.
• W2049366846 cites W2044707953 @default.
• W2049366846 cites W2045085957 @default.
• W2049366846 cites W2046559012 @default.
• W2049366846 cites W2046916908 @default.
• W2049366846 cites W2051886358 @default.
• W2049366846 cites W2056814797 @default.
• W2049366846 cites W2066430727 @default.
• W2049366846 cites W2070048444 @default.
• W2049366846 cites W2071756845 @default.
• W2049366846 cites W2071921711 @default.
• W2049366846 cites W2087922339 @default.
• W2049366846 cites W2092635505 @default.
• W2049366846 cites W2093491661 @default.
• W2049366846 cites W2094633691 @default.
• W2049366846 cites W2095174116 @default.
• W2049366846 cites W2104366258 @default.
• W2049366846 cites W2111537171 @default.
• W2049366846 cites W2116830452 @default.
• W2049366846 cites W2118842346 @default.
• W2049366846 cites W2133593669 @default.
• W2049366846 cites W2150543772 @default.
• W2049366846 cites W2152732725 @default.
• W2049366846 cites W2155185808 @default.
• W2049366846 cites W4210979710 @default.
• W2049366846 doi "https://doi.org/10.1074/jbc.m900985200" @default.
• W2049366846 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2682893" @default.
• W2049366846 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19304986" @default.
• W2049366846 hasPublicationYear "2009" @default.
• W2049366846 type Work @default.
• W2049366846 sameAs 2049366846 @default.
• W2049366846 citedByCount "56" @default.
• W2049366846 countsByYear W20493668462012 @default.
• W2049366846 countsByYear W20493668462013 @default.
• W2049366846 countsByYear W20493668462014 @default.
• W2049366846 countsByYear W20493668462015 @default.
• W2049366846 countsByYear W20493668462016 @default.
• W2049366846 countsByYear W20493668462017 @default.
• W2049366846 countsByYear W20493668462018 @default.
• W2049366846 countsByYear W20493668462019 @default.
• W2049366846 countsByYear W20493668462020 @default.
• W2049366846 countsByYear W20493668462021 @default.
• W2049366846 countsByYear W20493668462022 @default.
• W2049366846 crossrefType "journal-article" @default.
• W2049366846 hasAuthorship W2049366846A5000303187 @default.
• W2049366846 hasAuthorship W2049366846A5032745219 @default.
• W2049366846 hasAuthorship W2049366846A5064178833 @default.
• W2049366846 hasAuthorship W2049366846A5065229498 @default.
• W2049366846 hasAuthorship W2049366846A5066255279 @default.
• W2049366846 hasAuthorship W2049366846A5086468930 @default.
• W2049366846 hasBestOaLocation W20493668461 @default.
• W2049366846 hasConcept C170493617 @default.
• W2049366846 hasConcept C178790620 @default.
• W2049366846 hasConcept C185592680 @default.
• W2049366846 hasConcept C18903297 @default.
• W2049366846 hasConcept C2777289219 @default.
• W2049366846 hasConcept C28859421 @default.
• W2049366846 hasConcept C48349386 @default.
• W2049366846 hasConcept C540031477 @default.
• W2049366846 hasConcept C55493867 @default.
• W2049366846 hasConcept C61174792 @default.

• next