Control of glutamate oxidation in brain and liver mitochondrial systems

1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the α-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1-¹⁴C]- and [5-¹⁴C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of α-oxoglutarate or after decarboxylation to form γ-aminobutyrate. There is no indication of γ-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD⁺, the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the `free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields α-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is present in great excess compared with the respiration rate, the oxaloacetate formed is continuously removed by the transamination reaction. Thus α-oxoglutarate is formed independently of glutamate dehydrogenation, and the question is how the dehydrogenation of glutamate is influenced by the continuous formation of α-oxoglutarate. The results indicate that a competition takes place between the α-oxoglutarate-dehydrogenase complex and glutamate dehydrogenase, probably for NAD⁺, resulting in preferential oxidation of α-oxoglutarate.     

Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. II. Significance of binding of glutamate dehydrogenase to microsomal membrane

1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.     

Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N

We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK(1)-F(+) cell line. We used high-affinity glutamate transport inhibitors D-aspartate (D-Asp) and DL-threo-beta-hydroxyaspartate (THA) to block extracellular uptake and then used [(15)N]glutamate or [2-(15)N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-(15)N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mM D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% (P < 0.05) as the extracellular enrichment increased by 39% (P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% (P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH formed from [2-(15)N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both D-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.     

Detection of structural differences between nuclear and mitochondrial glutamate dehydrogenases by the use of immunoadsorbents.

Structural differences between crystalline mitochondrial and nuclear glutamate dehydrogenases from ox liver have been detected by immunological techniques. Antisera prepared against each enzyme precipitate both glutamate dehydrogenases; upon immunodiffusion, the antiserum against the nuclear enzyme gives a line of incomplete identity with the two antigens, whereas the antiserum against the mitochondrial enzyme gives a line of complete identity. Fractionation of the antibodies contained in each antiserum by means of an immunoadsorbent, to which the nuclear or the mitochondrial enzyme has been covalently linked, shows that nuclear glutamate dehydrogenase (GDH) contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, whereas the latter appears to have no antigenic portions which are not present in the nuclear antigen, in accord with the results of immunodiffusion. The antibodies against determinants common to both enzymes precipitate and inhibit them, whereas the specific anti-nuclear GDH antibodies precipitate but do not inhibit the nuclear antigen.     

Oligodendrocyte excitotoxicity determined by local glutamate accumulation and mitochondrial function

Developing oligodendrocytes (OL precursors, pre‐OLs) express α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) subtype glutamate receptors (AMPARs) and are highly vulnerable to hypoxic‐ischemic or oxygen‐glucose deprivation (OGD)‐induced excitotoxic injury, yet the mechanisms of injury remain unclear. Here we investigated the role of glutamate accumulation and mitochondrial function in OGD‐induced pre‐OL toxicity in vitro. Bulk glutamate concentration in the culture medium did not increase during OGD and OGD‐conditioned medium did not transfer toxicity to naïve cells. Facilitation of glutamate diffusion by constant agitation of the culture reduced, while inhibition of glutamate diffusion by increasing medium viscosity with dextran enhanced, OGD‐induced pre‐OL injury. Depletion of extracellular glutamate by the glutamate scavenging system, glutamate‐pyruvate transaminase plus pyruvate, attenuated pre‐OL injury during OGD. Together these data suggest that local glutamate accumulation is critical for OGD toxicity. Interestingly, under normoxic conditions, addition of glutamate to pre‐OLs did not cause receptor‐mediated toxicity, but the toxicity could be unmasked by mitochondrial impairment with mitochondrial toxins. Furthermore, OGD caused mitochondrial potential collapse that was independent of AMPAR activation, and OGD toxicity was enhanced by mitochondrial toxins. These data demonstrate that pre‐OL excitotoxicity is exacerbated by mitochondrial dysfunction during OGD. Overall, our results indicate that OGD‐induced pre‐OL injury is a novel form of excitotoxicity caused by the combination of local glutamate accumulation that occurs without an increase in bulk glutamate concentration and mitochondrial dysfunction. Therapeutic strategies targeting local glutamate concentration and mitochondrial injury during hypoxia‐ischemia may be relevant to human disorders associated with pre‐OL excitotoxicity.     

Impaired mitochondrial glutamate transport in autosomal recessive neonatal myoclonic epilepsy

Severe neonatal epilepsies with suppression-burst pattern are epileptic syndromes with either neonatal onset or onset during the first months of life. These disorders are characterized by a typical electroencephalogram pattern--namely, suppression burst, in which higher-voltage bursts of slow waves mixed with multifocal spikes alternate with isoelectric suppression phases. Here, we report the genetic mapping of an autosomal recessive form of this condition to chromosome 11p15.5 and the identification of a missense mutation (p.Pro206Leu) in the gene encoding one of the two mitochondrial glutamate/H(+) symporters (SLC25A22, also known as "GC1"). The mutation cosegregated with the disease and altered a highly conserved amino acid. Functional analyses showed that glutamate oxidation in cultured skin fibroblasts from patients was strongly defective. Further studies in reconstituted proteoliposomes showed defective [(14)C]glutamate uniport and [(14)C]glutamate/glutamate exchange by mutant protein. Moreover, expression studies showed that, during human development, SLC25A22 is specifically expressed in the brain, within territories proposed to contribute to the genesis and control of myoclonic seizures. These findings provide the first direct molecular link between glutamate mitochondrial metabolism and myoclonic epilepsy and suggest potential insights into the pathophysiological bases of severe neonatal epilepsies with suppression-burst pattern.     

Mechanisms underlying the loss of mitochondrial membrane potential in glutamate excitotoxicity

Glutamate excitotoxicity amplifies neuronal death following stroke. We have explored the mechanisms underlying the collapse of mitochondrial potential (Deltapsi(m)) and loss of [Ca(2+)](c) homeostasis in rat hippocampal neurons in culture following toxic glutamate exposure. The collapse of Deltapsi(m) is multiphasic and Ca(2+)-dependent. Glutamate induced a decrease in NADH autofluorescence which preceded the loss of Deltapsi(m). Both the decrease in NADH signal and the loss of Deltapsi(m) were suppressed by Ru360 and both were delayed by inhibition of PARP (by 3-AB or DPQ). During this period, addition of mitochondrial substrates (methyl succinate and TMPD-ascorbate) or buffering [Ca(2+)](i) (using BAPTA-AM or EGTA-AM), rescued Deltapsi(m). These data suggest that mitochondrial Ca(2+) uptake activates PARP which in turn depletes NADH, promoting the initial collapse of Deltapsi(m). After > approximately 20 min, buffering Ca(2+) or substrate addition failed to restore Deltapsi(m). In neurons from cyclophilin D-/- (cypD-/-) mice or in cells treated with cyclosporine A, removal of Ca(2+) restored Deltapsi(m) even after 20 min of glutamate exposure, suggesting involvement of the mPTP in the irreversible depolarisation seen in WT cells. Thus, mitochondrial depolarisation represents two consecutive but distinct processes driving cell death, the first of which is reversible while the second is not.     

An artifact in the radiochemical assay of brain mitochondrial glutamate decarboxylase

Commercial DL-[1-¹⁴C] glutamic acid contains an impurity from which ¹⁴CO₂ is released during incubation with brain mitochondrial glutamate decarboxylase and the inhibitor aminooxyacetic acid. This results in an apparent stimulation of brain mitochondrial glutamate decarboxylase by aminooxyacetic acid when low levels of the enzyme are used. Both aminooxyacetic acid and chloride ion inhibited both the supernatant and mitochondrial glutamate decarboxylase activities when purified DL-[1-¹⁴C] glutamic acid was used as substrate.     

The precursor of rat liver mitochondrial glutamate dehydrogenase has enzymatic activity

The cytosolic precursor for the mitochondrial glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using messenger RNA from rat liver. To check whether this precursor had enzymatic activity, a highly sensitive fluorimetric method, which can measure picogram quantities of enzyme, was used together with competitive dissociation of the precursor from an immunoprecipitate with inactive glutamate dehydrogenase. Glutamate dehydrogenase activity, corresponding to that estimated from incorporation of [35S]-methionine, was detected in the precursor. The significance of this finding is discussed.