Kim Ping Wong

A Mechanism of Sulfite Neurotoxicity DIRECT INHIBITION OF GLUTAMATE DEHYDROGENASE

Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 μm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in α-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants.

Gas chromatographic measurement of urinary catecholamines by an electron capture detection procedure

Abstract A gas-liquid Chromatographic procedure with electron capture detection has been developed to measure the catecholamines, noradrenaline, adrenaline and dopamine in normal human urine as their pentafluoropropionyl derivatives. The amines are absorbed from urine at pH 8.5 on micro-columns of alumina, eluted with acetic acid and derivatized with pentafluoropropionic anhydride. Some improvements in the conventional procedure of chromatography on alumina have been introduced. Quantification is obtained by comparing peak heights with those of internal standards added to urine and taken through the entire determination. Normal urinary excretion values of free catecholamines (mean ± S.D.), expressed as μg per 24 h, were: noradrenaline, 52.1 ± 15.0, adrenaline, 24.2 ± 4.0 and dopamine 402.4 ± 86.3. Conjugated dopamine can be determined after acid hydrolysis (pH 1, 100°, 20 min). About 50 to 75% of total dopamine output is excreted in conjugated form.

Do Mitochondria make Nitric Oxide? No?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO•) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of l-arginine or the NOS inhibitor l-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO• production by the oxymyoglobin oxidation assay, or electrochemically using an NO• electrode. An apparent NO• production detected by the Griess assay was identified as an artifact. NO• generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO•. Our data show that NO• production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.

PTEN/Akt signaling controls mitochondrial respiratory capacity through 4E-BP1.

Akt, a serine/threonine kinase has been shown to stimulate glycolysis in cancer cells but its role in mitochondrial respiration is unknown. Using PTEN-knockout mouse embryonic fibroblasts (MEFPTEN−/−) with hyper-activated Akt as a cell model, we observed a higher respiratory capacity in MEFPTEN−/− compared to the wildtype (MEFWT). The respiratory phenotype observed in MEFPTEN−/− was reproduced in MEFWT by gene silencing of PTEN which substantiated its role in regulating mitochondrial function. The increased activities of the respiratory complexes (RCs) I, III and IV were retained in the same relative proportions as those present in MEFWT, alluding to a possible co-ordinated regulation by PTEN/Akt. Using LY294002 (a PI3K inhibitor) and Akt inhibitor IV, we showed that the regulation of enzyme activities and protein expressions of the RCs was dependent on PI3K/Akt. There was insignificant difference in the protein expressions of mitochondrial transcription factor: peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and its downstream targets, the nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (mtTFA) between MEFPTEN−/− and MEFWT. Similarly, mRNA levels of the same subunits of the RCs detected in Western blots were not significantly different between MEFPTEN−/− and MEFWT suggesting that the regulation by Akt on mitochondrial function was probably not via gene transcription. On the other hand, a decrease of total 4E-BP1 with a higher expression of its phosphorylated form relative to total 4E-BP1 was found in MEFPTEN−/−, which inferred that the regulation of mitochondrial respiratory activities by Akt was in part through this protein translation pathway. Notably, gene silencing of 4E-BP1 up-regulated the protein expressions of all RCs and the action of 4E-BP1 appeared to be specific to these mitochondrial proteins. In conclusion, PTEN inactivation bestowed a bioenergetic advantage to the cells by up-regulating mitochondrial respiratory capacity through the 4E-BP1-mediated protein translation pathway.

Sulphite oxidase gene expression in human brain and in other human and rat tissues.

Sulphite oxidase (EC 1.8.3.1) is a molybdopterin-containing enzyme that catalyses the oxidation of sulphite to sulphate. Lack of active enzyme produces severe neurodegeneration and early death in humans, showing its essential role. Despite this, the expression of the sulphite oxidase gene in human and rat tissues (especially the brain) has not been elucidated. We therefore examined these tissues and found that the human liver, kidney, skeletal muscle, heart, placenta, and brain showed substantial expression while thymus, spleen, peripheral blood leucocytes, colon, small intestine, and lung showed little expression in humans. In rat, the liver, kidney, heart, brain, and lung (but not skeletal muscle) revealed a hybridization signal with the strongest signal in the liver. The spleen and testis also showed little expression. The differential expression of sulphite oxidase gene in various human brain regions was studied. Expression was seen in all brain regions examined (cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, amygdala, caudate nucleus, corpus callosum, hippocampus, thalamus, temporal lobe, and putamen). The cerebral cortex showed the highest level of expression.

Lactacystin-induced apoptosis of cultured mouse cortical neurons is associated with accumulation of PTEN in the detergent-resistant membrane fraction

AbstractThe tumor suppressor function of PTEN is attributed to its phospholipid phosphatase activity that dephosphorylates the plasma membrane phosphatidylinositol-(3,4,5)-triphosphate [PtdIns(3,4,5)P3]. Implicit in this notion is that PTEN needs to be targeted to the plasma membrane to dephosphorylate PtdIns(3,4,5)P3. However, the recruitment of PTEN to the plasma membrane is not fully understood. Here, we demonstrate PTEN accumulation in the detergent-insoluble fraction of neuronal cells in response to treatment by the proteasome inhibitor lactacystin. First, lactacystin induces apoptosis and the activation of caspase-3 in cultured cortical neurons. Second, PTEN undergoes proteolysis to form a truncated 50-kDa form that lacks parts of its C-terminal tail. Third, the truncated PTEN is stably associated with the detergent-insoluble fraction in which the plasma membrane marker protein flotillin-1 resides. Taken together, our results suggest that truncation and accumulation of PTEN to the detergent- insoluble membrane fraction are two events associated with the apoptotic signals of the proteasome inhibitor in cortical neurons.

Respiratory competent mitochondria in human ovarian and peritoneal cancer.

Impaired respiration was proposed by Warburg to be responsible for aerobic glycolysis in cancer cells. However, intact mitochondria isolated from human ovarian and peritoneal cancer tissues exhibit substantive oxidative phosphorylating activities in terms of membrane potential, ATP biosynthesis and oxygen consumption. The specific activities of succinate, malate and glutamate dehydrogenases are comparable to reported values for human skeletal muscle, heart and liver but the rate of ATP production is one order of magnitude lower compared to human skeletal muscle. It was concluded that the TCA cycle is functional in these ovarian cancer tissues which contain OXPHOS competent mitochondria.

Nephrotoxic cell death by diclofenac and meloxicam

The nephrotoxicity of diclofenac, a non-steroidal anti-inflammatory drug that inhibits both isoforms of cyclooxygenase (COX) has been reported to be fatal to vultures but this was not so with meloxicam which is COX-2 selective. Our study showed that diclofenac was more toxic than meloxicam to both the proximal tubular LLC-PK1 cells and the distal tubular Madin-Darby canine kidney type II (MDCKII) cells, and that LLC-PK1 cells were more susceptible. Exposure of MDCKII cells to meloxicam caused activation of caspase-9/-3 and release of cytochrome c. These observations together with a positive annexin V-FITC staining implicate an intrinsic mitochondrial cell death pathway by apoptosis. Diclofenac-treated MDCKII cells on the other hand showed extensive propidium iodide staining, suggestive of cell death by necrosis. The mode of cell death in LLC-PK1 cells was however less well-defined with positive annexin V-FITC staining but minimal increase in caspase-3 activity alluding to a caspase-independent pathway.

Antiport-mediated cell retraction: viable rounding and distinctive endocytosis

Cells that are detached through Na+/H+ antiport-mediated cell retraction (the flat-to-round or FTR change) are as viable as trypsinized cells despite unusually high trypan blue dye inclusion. Even high molecular weight dextran and carbon particles are easily loaded following the FTR change. ECM of confluent cultures of human keloid fibroblasts and Chang liver epitheloid cells stained directly by fluorescein-conjugated monoclonal anti-fibronectin is dramatically reduced or removed upon cell rounding. At the ultrastructural level, distinctive channels and vacuoles filled with extracellular material are seen, resolved at later stages as peripheral granular patches which too disappear when the rounded cells are reflattened in round-to-flat (RTF) change. Antiport-mediated endocytosis (AME) could explain the drastic reduction in cell area concomitant with cell rounding and detachment. AME provides a new way of loading cells with impermeant macromolecules.

N-acetylation of dopamine and tyramine by mosquito pupae (Aedes togoi)

Abstract The N- acetyltransferase (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [ 14 C]-, [ 3 H]dopamine, [ 14 C]tyramine or [ 14 C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn 2+ stimulated and Zn 2+ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, 893 ± 57 and 861 ± 30 pmol [ 14 C]NAcT formed/min per mg protein and 21.9 ± 1.2 and 22.0 ± 1.4 pmol [ 3 H]NADA formed/min per mg protein.