James C. K. Lai

Brain alpha-ketoglutarate dehydrogenase complex : kinetic properties, regional distribution, and effects of inhibitors

Abstract The substrate and cofactor requirements and some kinetic properties of the α‐ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for α‐ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex > cerebellum ≧ midbrain > striatum = hippocampus > hypothalamus > pons and medulla > olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2–2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both β‐methylene‐D,L‐aspartate and D,L‐vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 μM, calcium slightly stimulated (by 10–15%) the brain mitochondrial KGDHC.

Inhibition of Brain Glycolysis by Aluminum

Abstract: Aluminum inhibited both the cytosolic and mitochondrial hexokinase activities in rat brain. The IC50 values were between 4 and 9 μM. Aluminum was effective at mildly acidic (pH 6.8) or slightly alkaline (pH 7.2–7.5) pH, in the presence of a physiological level of magnesium (0.5 mM). However, saturating (8 mM) magnesium antagonized the effect of aluminum on both forms of hexokinase activity. Other enzymes examined were considerably less sensitive to inhibition by aluminum. The IC50 of aluminum for phosphofructokinase was 1.8 mM and for lactate dehydrogenase 0.4 mM. At 10–600 μM, aluminum actually stimulated pyruvate kinase. Aluminum also inhibited lactate production by rat brain extracts: this effect was much more marked with glucose as substrate than with glucose‐6‐phosphate. However, the IC50 for inhibiting lactate production using glucose as substrate was 280 μM, higher than that required to inhibit hexokinase. This concentration of aluminum is comparable to those reportedly found in the brains of patients who had died with dialysis dementia and in the brains of some of the patients who had died with Alzheimer disease. Inhibition of carbohydrate utilization may be one of the mechanisms by which aluminum can act as a neurotoxin.

Cerebral ammonia metabolism in normal and hyperammonemic rats

Brain ammonia is generated from many enzymatic reactions, including glutaminase, glutamate dehydrogenase, and the purine nucleotide cycle. In contrast, the brain possesses only one major enzyme for the removal of exogenous ammonia, i.e., glutamine synthetase. Thus, following administration of [13N]ammonia to rats [via either the carotid artery or cerebrospinal fluid (csf)], most metabolized label was in glutamine (amide) and little was in glutamate (plus aspartate). Since blood-and csf-borne ammonia are converted to glutamine largely, if not entirely, in the astrocytes, it is not possible from these types of experiments to predict with certainty the metabolic fate of the bulk of endogenously produced ammonia. By comparing the specific activity of L-[13N]glutamate to that of L-[amine-13N]glutamine following intracarotid [13N]ammonia administration it was concluded that metabolic compartmentation is no longer intact in the brains of rats treated with the glutamine synthetase inhibitor L-methionine-SR-sulfoximine (MSO) and that blood and brain ammonia pools mix in such animals. In MSO-treated animals, recovery of label in brain was low (approximately 20% of controls), and of the label remaining, a prominent portion was in glutamine (amide) (despite an 87% decrease in brain glutamine synthetase activity). These data are consistent with the hypothesis that glutamine synthetase is the major enzyme for metabolism of endogenously--as well as exogenously--produced ammonia. The rate of turnover of blood-derived ammonia to glutamine in normal rat brain is extremely rapid (t1/2 less than or equal to 3 s), but is slowed in the brains of chronically (12-14-wk portacaval-shunted) or acutely (urease-treated) hyperammonemic rats (t1/2 less than or equal to 10 s). The slowed turnover rate may be caused by an increased astrocytic ammonia, decreased glutamine synthetase activity, or both. In the hyperammonemic rat brain, glutamine synthetase is still the only important enzyme for the removal of blood-borne ammonia. Hyperammonemia causes an increase in brain lactate/pyruvate ratios and decreases in brain glutamate and brainstem ATP, consistent with an interference with the malate-aspartate shuttle. In vitro, pathological levels of ammonia also inhibit brain alpha-ketoglutarate dehydrogenase complex and, less strongly, pyruvate dehydrogenase complex. The rat brain does not adapt to prolonged hyperammonemia by increasing its glutamine synthetase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of ammonia and β-methylene-DL-aspartate on metabolism of glutamate and related amino acids by astrocytes and neurons in primary culture

The effects of ammonium chloride (3 mM) and β-methylene-dl-aspartate (BMA; 5 mM) (an inhibitor of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle (MAS)) on the metabolism of glutamate and related amino acids were studied in primary cultures of astrocytes and neurons. Both ammonia and BMA inhibited14CO2 production from [U-14C]-and [1-14C]glutamate by astrocytes and neurons and their effects were partially additive. Acute treatment of astrocytes with ammonia (but not BMA) increased astrocytic glutamine. Acute treatment of astrocytes with ammonia or BMA decreased astrocytic glutamate and aspartate (both are key components of the MAS). Acute treatment of neurons with ammonia decreased neuronal aspartate and glutamine and did not apparently affect the efflux of aspartate from neurons. However, acute BMA treatment of neurons led to decreased neuronal glutamate and glutamine and apparently reduced the efflux of aspartate and glutamine from neurons. The data are consistent with the notion that both ammonia and BMA may inhibit the MAS although BMA may also directly inhibit cellular glutamate uptake. Additionally, these results also suggest that ammonia and BMA exert differential effects on astroglial and neuronal glutamate metabolism.

Neurotoxicity of ammonia and fatty acids: Differential inhibition of mitochondrial dehydrogenases by ammonia and fatty acyl coenzyme a derivatives

In several metabolic encephalopathies, hyperammonemia and organic acidemia are consistently found. Ammonia and fatty acids (FAs) are neurotoxic: previous workers have shown that ammonia and FAs can act singly, in combination, or synergistically, in inducing coma in experimental animals. However, the biochemical mechanisms underlying the neurotoxicity of ammonia and FAs have not been fully elucidated. FAs are normally converted to their corresponding CoA derivatives (CoAs) once they enter cells and it is known that these fatty acyl CoAs can alter intermediary metabolism. The present study was initiated to determine the effects of ammonia and fatty acyl CoAs on brain mitochondrial dehydrogenases. At a pathophysiological level (2 mM), ammonia is a potent inhibitor of brain mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC). Only at toxicological levels (10–20 mM) does ammonia inhibit brain mitochondrial NAD+- and NADP+-linked isocitrate dehydrogenase (NAD-ICDH, NADP-ICDH), and NAD+-linked malate dehydrogenase (MDH) and liver mitochondrial NAD-ICDH. Butyryl- (BCoA), octanoyl- (OCoA), and palmitoyl (PCoA) CoA were potent inhibitors of brain mitochondrial KGDHC, with IC50 values of 11, 20, and 25 μM, respectively; moreover, the inhibitory effect of fatty acyl CoAs and ammonia were additive. At levels of 250 μM or higher, both OCoA (IC50=1.15 mM) and PCoA (IC50=470 μM) inhibit brain mitochondrial NADP-ICDH; only at higher levels (0.5–1 mM) does BCoA inhibit this enzyme (by 30–45%). Much less sensitive than KGDHC and NADP-ICDH, brain mitochondrial NAD-ICDH is only inhibited by 1 mM BCoA, OCoA, and PCoA by 22%, 35%, and 44%, respectively. Even at 1 mM, OCoA and PCoA (but not BCoA) only slightly inhibited brain mitochondrial MDH (by 23%). In the presence of toxicological levels of ammonia (20 mM) and fatty acyl CoAs (1 mM), the inhibitory effect of fatty acyl CoAs and ammonia on brain mitochondrial NAD-ICDH, NADP-ICDH, and MDH are only partially additive. These results provide some support for our hypothesis that selective inhibition of a rate-limiting and regulated enzymatic step (e.g., KGDHC) by ammonia and fatty acyl CoAs may be one of the major mechanisms underlying the neurotoxicity of ammonia and FAs. The data also suggest that the same mechanism may acocunt for the synergistic effect of ammonia and FAs in inducing coma. Since the inhibition of KGDHC by ammonia and fatty acyl CoAs occurs at pathophysiological levels, the results may assume some pathophysiological and/or pathogenetic importance in metabolic encephalopathies in which hyperammonemia and organic acidemia are persistent features.

Some metabolic effects of ammonia on astrocytes and neurons in primary cultures

Some metabolic effects on primary cultures of neurons or astrocytes were studied following acute or chronic exposure to pathophysiological concentrations (usually 3 mM) of ammonia. Three parameters were investigated: (1) 14CO2 production from 14C-labeled substrates [glucose, pyruvate, branched-chain amino acids (leucine, valine, isoleucine), and glutamate]; (2) interconversion between glutamate and glutamine; and (3) incorporation of label from labeled branched-chain amino acids into proteins. Neither acute nor chronic exposure to ammonia had any effect on 14CO2 production from [U-14C]glucose in astrocytes and neurons, whereas under certain conditions 14CO2 production from [1-14C]pyruvate in astrocytes was inhibited by ammonia. Production of 14CO2 from [1-14C]branched-chain amino acids was inhibited by acute, but stimulated by chronic, exposure to ammonia (3 mM) in astrocytes, with less effect in neurons. Production of 14CO2 from [1-14C]glutamate in both astrocytes and neurons was inhibited by acute exposure to ammonia. In astrocytes, glutamate levels tended to decrease and glutamine levels tended to increase following acute exposure to ammonia; in neurons, both glutamine and glutamate levels decreased. Protein content (per culture dish) increased in astrocytes but not in neurons, after chronic exposure to ammonia, possibly as a result of enhanced protein synthesis and/or by inhibition of protein degradation.

Pyruvate dehydrogenase complex is inhibited in calcium-loaded cerebrocortical mitochondria

An impairment of mitochondrial functions as a result of Ca-loading may be one of the significant events that lead to neuronal death after an ischemic insult. To assess the metabolic consequences of excess Ca on brain mitochondria, pyruvate oxidation was studied in isolated cerebrocortical mitochondria loaded with Ca in vitro. The flux of pyruvate dehydrogenase complex (PDHC) ([1-14C]pyruvate decarboxylation) was inhibited as the mitochondria accumulated excess Ca under the conditions tested: the inhibition in state 3 (i.e., in the presence of added ADP) was greater than in state 4 (i.e., in the absence of added adenine nucleotides). In state 4, the inhibition of the PDHC flux was accompanied by a similar reduction of the in situ activity of PDHC, indicating a change in PDHC phosphorylation. In state 3, the inhibition of the PDHC flux was greater than the corresponding decrease of the in situ PDHC activity. Thus, mechanisms other than the phosphorylation of PDHC might also contribute to the inhibition of pyruvate oxidation. Measurement of PDHC enzymatic activity in vitro indicated that PDHC, similar to α-ketoglutarate dehydrogenase complex, was inhibited by millimolar levels of Ca. This observation suggests that PDHC may also be inhibited non-covalently in Ca-loaded mitochondria in a manner similar to that of α-ketoglutarate dehydrogenase complex.

An application of neutron activation analysis to small biological samples: simultaneous determination of thirty elements in rat brain regions.

Thirty elements in 7 rat brain regions were determined by instrumental neutron activation analysis (INAA). The samples were irradiated by thermal neutrons using 3 different sets of conditions, depending on the nuclear characteristics of the elements. Analysis of the resulting radionuclides was by gamma-ray spectrometry using a high resolution Ge(Li) detector and Nuclear Data 6600 multichannel analyzer, which was fully computerized to give quantitative results for the gamma-ray spectra. This paper demonstrates the use of INAA for small biological samples and to show its potential elements, 7 rat brain regions are listed. It is interesting to note that certain elements, e.g. fluorine and potassium showed high and low regional differences respectively, and hypothalamus and hippocampus had higher elemental concentrations than other brain regions. However, since this paper is essentially an analytical one, no attempt is made to assess these data, which are preliminary, and the possible functional role of these elements will be discussed elsewhere.

Pyruvate Dehydrogenase Phosphate (PDHb) Phosphatase in Brain: Activity, Properties, and Subcellular Localization

Abstract: The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg·ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho‐PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K‐phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K‐phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial faction, while the activities of other “mitochondrial” enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubizeded only by elevated concentrations of detergents (0.3% each) plus KCl (>150mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.